Research Collection

Doctoral Thesis

Environmental factors influencing ecological interactionsbetween biocontrol Pseudomonads and fungal pathogens

Author(s): Duffy, Brion

Publication Date: 1999

Permanent Link: https://doi.org/10.3929/ethz-a-002063756

Rights / License: In Copyright - Non-Commercial Use Permitted

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ETH Library

Diss ETH Nr. 13023

Environmental factors influencing ecological interactions

between biocontrol pseudomonads and fungal pathogens

A dissertation for the degree of Doctor of Technical Sciences submitted to the

Swiss Federal Institute of Technology, Zürich

Presented by Brion DUFFY

BSc Crop Protection, University of Hawaii at Hilo

MSc Plant Pathology, Washington State UniversityBorn 21 August 1967 USA

Accepted on the recommendation of:

Prof. Dr. Geneviève Défago, referent

Prof. Dr. Emmannuel Frossard. co-referent

Dr. David M. Weiler, co-referent

1999 / A/.y.

/y <". 9'/

Contents

Abbreviations

General Summary 1

Résumé Général 3

Background 5

Chapter 1. 13

Mineral amendments reduce the accumulation of spontaneous gacS-gacA regulatory

mutants during liquid culture of Pseudomonas fluorescens biocontrol strains

Chapter 2. 41

Environmental factors modulating antibiotic and siderophore production by

Pseudomonasfluorescens biocontrol strains (Appl Environ Microbiol, accepted)

Chapter 3. 71

Zinc improves biocontrol of Fusarium crown and root rot of tomato by

Pseudomonas fluorescens and represses the production of pathogen metabolites

inhibitory to bacterial antibiotic biosynthesis (Phytopathology 87:1250-1257)

Chapter 4. 95

A Fusarium pathogenicity factor blocks antibiotic biosynthesis by antagonistic

pseudomonads (IOBCwprs Bulletin 21(9): 145-148)

Chapter 5. 101

Macro- and microelements influence the severity of Fusarium crown and root rot

of tomato in a soilless production system (HortScience, in press)

General Conclusions 117

Acknowledgements 119

Curriculum vitae 121

Publications 123

Abbreviations

ARDRA

CFU

Cm

EDDHA

EDTA

FA

FORL

gac

GNB

HCN

HPLC

KB

NB

NBY

PCG

PCR-RAPDs

PHL

PUT

Rif

SAL

SAS

TSO

amplified ribosomal DNA restriction analysis

colony-forming unit

chloramphenicol

ethylcnediamine-di(o-hydroxyphcnyl-acetic acid)

ethylenediaminetetraaceticacicl

fusaric acid

Fusarium oxysporum f.sp. radicis-lycopersici

global activator of antibiotic and cyanide biosynthesis

gelatin-nutrient broth medium

hydrogen cyanide

high-performance liquid chromatography

King's medium B

least significant difference test

nutrient broth medium

nutrient broth-yeast extract medium

peptone-casamino acids glucose medium

polymerase chain reaction based fingerprinting with randomly

amplified polymorphic DNA markers

2,4-diacetylphloroglucinol

pyoluteorin

rifampicin

salicylic acid

statistical analysis systems of the SAS Institute

tryptophan side-chain oxidase

1

General Summary

Biocontrol using beneficial Pseudomonas fluorescens is one of the most promising approaches

to manage soilborne diseases, for which agrochemicals are generally ineffective. Variable

strain performance, however, has hampered commercialization efforts. With the aim of

overcoming this problem, this thesis identified factors that directly and indirectly influence the

level and reliability of biocontrol.

• Genetic stability and minerals: High frequency (>1%) spontaneous mutation in gacS-

gacA global regulators abolished antibiotic production and reduced the biocontrol efficacy

of Pseudomonas inoculants against Pythium damping-off of cucumber. Mutants had a

distinct colony appearance (ie, dark, flat, transparant, hyperfluorescent). Mutants were

favored in nutrient/electrolyte rich media. Trace minerals added to media (Zn, Co, Cu, Mn,

NH4Mo) improved genetic stability in strains from Switzerland, Ghana, and Italy.

» Minerals and antibiotic biosynthesis: Trace minerals and carbon sources modulated

biosynthesis of antimicrobial compounds in genetically and ecologically diverse biocontrol

strains. In the model Swiss strain Pseudomonas fluorescens CHAO, Zn stimulated the

antibiotics 2,4-diacetylphloroglucinol (PHL) and pyoluteorin (PLT), while glucose

stimulated PHL but repressed PLT. A mixture of Zn + NH,Mo with various carbon sources

further enhanced antibiotic production. Zn and glucose had the same effect on all strains

genetically closest to CHAO (ARDRA group 1), but the effect was strain dependent in other

ARDRA groups. Inorganic phosphate repressed PHL and PLT but not pyrrolnitrin.

* Pathogen signals, minerals and biocontrol: Fusaric acid is a Fusarium oxvsporum

phytotoxic pathogenicity factor. Here it was also found to block biosynthesis of PHL by

biocontrol Pseudomonas fluorescens, the first example of molecular signalling between

pathogens and antagonistic microbes. Fusaric acid also repressed PLT but did not affect

hydrogen cyanide and protease suggesting it acted downstream of gacS-gacA. In a soilless

rockwool system, the biocontrol activity of CHAO against Fusarium crown and root rot of

tomato was improved by 25% with Zn or Cu amendments (33 mg/L). Cu was fungttoxic,

but Zn worked via a less direct mechanism. Zn did not directly stimulate PHL production by

CHAO in situ as anticipated from the above in vitro studies. Rather, Zn repressed fusaric

acid production by the pathogen. Thus, Zn created a 'fusaric acid free-zone' where CHAO

produced PHL and was able to suppress the pathogen. Genetic analysis indicated that the

moderate level of biocontrol observed with CHAO in the presence of fusaric acid was

largely due to HCN production.

2

An ecologically distinct collection of strains was then tested for sensitivity to fusaric acid in

vitro. This pathogen signal blocked PHL production in all strains genetically related to

CHAO (PHL and PLT biosynthetic genes), but had no effect on PHL production by

genetically distinct strains (only PHL biosynthetic genes). Biocontrol of Fusarium crown

and root rot of tomato was negatively correlated with sensitivity to fusaric acid. Thus,

strains selected for the ability to produce PHL in the presence of fusaric acid were more

effective. The primary importance of PHL in biocontrol of Fusarium was demonstrated by

the fact that in a fusaric acid-resistant strain (Q2-87) intemption of PHL genes abolished

disease suppression.

• Mineral non-target effects: Potential non-target effects must be considered before

manipulating crop mineral nutrition. Regression analysis indicated that Fusarium crown and

root rot of tomato was increased by ammonium-N, NaH,POt-H,0, Fe-EDDHA, MnS04,

MoO,, and ZnSO,-7H20. Low NH4NO, rates (39-79 mg N/liter) reduced disease, but this

effect was reversed as rates increased above 100 mg N/liter. The Zn concentration (33

mg/L) used above to improve biocontrol was the upper limit possible without agravating

disease. Fertilization with nitrate-N or CuSO(-H,0 reduced disease and could be exploited

for crown and root rot management. Non-target effects on other beneficial bacteria must

also be considered. All strains genetically similar to CHAO were relatively tolerant to 0.7

mM Zn-sulphate, whereas, growth of biocontrol strains in other ARDRA groups was

inhibited by concentrations above 0.2 mM.

This thesis documents for the first time the risks posed by genetic instability and negative

pathogen (fusaric acid) signals in biocontrol. Approaches to improve genetic stability, stimulate

antibiotic biosynthesis and enhance biocontrol, particularly using mineral amendments (eg,

zinc), were developed.

3

Résumé Général

L'utilisation de Pseudomonas spp fluorescents contre les maladies microbiennes d'origine

tellurique est une alternative prometteuse à la lutte chimique, peu efficace pour ce type de

maladies. Néanmoins, le manque de reproductibilité des effets bénéfiques de la bactérisation en

retarde la commercialisation. Le but de cette thèse était d'identifier certains des facteurs

environnementaux qui modulent l'efficacité de la lutte biologique.

• Stabilité génétique et élément-traces : Une haute fréquence (>1 %) de mutations des gènes

de régulation gacS-gacA a supprimé la production de substances antimicrobiennes. De plus,

elle a réduit l'efficacité des inoculants contre le Pythium ultimum sur concombre. Les

mutants forment des colonies plus sombres, plus transparentes et plus fluorescentes que les

souches sauvages. Les mutants sont favorisés par les milieux riches en nutriments ou en

electrolytes. L'addition d'éléments-traces (Zn, Co, Cu, Mn ou NH(Mo) aux milieux de

culture a amélioré la stabilité génétique des souches provenant de Suisse, du Ghana ou

d'Italie.

• Elément-traces et biosynthèse des substances antimicrobiennes : les éléments-traces et

les sources de carbone ont modulé la biosynthèse des substances antimicrobiennes chez des

souches de Pseudomonas fluorescens, génétiquement et écologiqucment diverses. Chez la

souche-modèle CHAÜ, le Zn a stimulé la production du 2.4-diacétylphloroglucinol (PHL) et

de la pyolutéorme (PLT), deux substances antimicrobiennes. Le glucose, quant à lui, a

stimulé la production de PHL mais réprimé celle de la PLT. Un mélange de Zn, de NH(Mo

et de diverses sources de carbone a augmenté encore davantage la production d'substances

antimicrobiennes. Le Zn et le glucose ont eu le même effet sur toutes les souches

génétiquement proches de CHAO (groupe ARDRA 1), mais l'effet était dépendant de la

souche dans d'autres groupes ARDRA. Le phosphate minéral a réprimé la biosynthèse du

PHL et de la PLT mais pas de la pyrrolnitrine.

• Signaux de l'agent pathogène, élément-traces et biocontrôle : l'acide fusarique, une

Phytotoxine du Fusarium owsporum. a bloqué la biosynthèse du PHL chez Pseudomonas

fluorescens CHAO. C'est le premier exemple d'un signal moléculaire entre un pathogène et

un agent de biocontrôle. L'acide fusarique a réprimé aussi la synthèse du PLT mais non

celle de l'HCN ni des proteases, suggérant que l'acide fusarique agit en aval de gacS-gacA.

Dans un système hors sol, l'apport de Zn (33 mg/L) ou de Cu a amélioré l'efficacité du

contrôle biologique par CHAO. L'analyse des solutions nutritives, à la fin de l'expérience, a

4

montré que le Cu avait inhibé la croissance du champignon. Le Zn, quant à lui, n'avait pas

inhibé la croissance du champignon ni augmenté la synthèse du PHL, ce que laissaient

supposer les expériences in vitro. Il avait agi indirectement, en réprimant la synthèse de

l'acide fusarique. Le Zn avait donc créé un espace sans acide fusarique, permettant ainsi à

CHAO de synthétiser du PHL et de protéger les plantes.

• Effets secondaires des élément-traces : l'apparition d'effets secondaires indésirables doit

être prise en considération avant de modifier la nutrition minérale des plantes. Une analyse

de régression a indiqué que la pourriture du collet et des racines de tomate causée par le F.

oxysporum f. sp. radias était augmentée par l'apport de NH4', de NaH2P04, Fe-FDDHA,

MnSOp MoO, ou ZnS04 .Des taux bas de NH4NO, (39-79 mg N/L) ont réduit le degré de

maladie, mais des taux élevés (plus de 100 mg N/L) ont eu un effet contraire. La

concentration de Zn (33 mg/L), utilisée pour améliorer l'efficacité du contrôle biologique,

était la concentration la plus élevée possible pour ne pas aggraver la maladie. La

fertilisation avec du Ca(N0„), ou du CuSOt a réduit l'intensité de la maladie et pourrait être

exploitée pour contrôler la pourriture du collet et des racines de tomate. Les effets

indésirables sur d'autres bactéries, agents de lutte biologique, doivent aussi être pris en

considération. Toutes les souches génétiquement proches de CHAO (groupe ARDRA 1) ont

poussé relativement bien en présence de 0.7 mM de sulfate de Zn, alors que les souches des

autres groupes ARDRA ont été inhibées par des concentrations supérieures à 0.2 mM.

Cette thèse documente, pour la première fois, les risques posés par l'instabilité génétique des

inoculants et par les signaux moléculaires négatifs (acide fusarique) de l'agent pathogène sur

l'efficacité de la lutte biologique. L'utilisation d'éléments-traces, en particulier du Zn, a permis

d'améliorer la stabilité génétique, de stimuler la synthèse de substances antimicrobiennes et

d'augmenter l'efficacité de la lutte biologique par des Pseudomonas fluorescens.

5

Background

Biological control in plant pathology usually refers to the introduction of various

nonpathogenic microorganisms (eg., fungi, bacteria, viruses) to suppress crop diseases

and to reduce postharvest losses. Biocontrol has in the past been most successfully used

to manage diseases for which other alternatives were unavailable (Cook 1993). The best

example is biocontrol of crown gall (caused by Agrobacteriwn tumefaciens) on woody

perennials using non-tumorigenic A. rhizogenes (formerly A. radiobacter) strain K84

and derivatives (McClure et al. 1998). Neither host resistance nor chemicals were

previously available, and biocontrol was the first real advance in crown gall control.

Increasingly though, biocontrol is also being called upon to replace agrochemicals in

'organic' or 'sustainable' farming systems, and to replace banned chemicals like

methyl-bromide which is being phased-out in the US and the EU by 2005 (Ristaino and

Thomas 1997). Despite great potential and growing demand for biocontrol,

disappointingly few products have been registered. As of 1997, only 5 fungal and 10

bacterial products were approved by the Environmental Protection Agency in the US,

only 3 were registered in The Netherlands, and none have been registered EU-wide.

Commercialization has been hampered by complicated, and therefore high-cost,

registration guidelines which discourage small-niche markets typical for biocontrol. and

lack-luster public support due in part to perceived risks associated with large-scale use

of biocontrol agents. Some relief has been achieved through regulatory modifications

(Cook 1993, Kenney 1997) and biosafety study (Natsch et al. 1997).

However, biological constraints on the disease suppressive activity of individual

strains remain and these must also be overcome before the full potential of biocontrol

can be realized (Weiler 1988). Key goals arc to optimize the level of protection and

expand the spectrum of diseases controlled, improve strain reliability from site to site

and from year to year, and develop cost-effective formulations that give consistent

results. Much of the work done to accomplish these goals has been with fluorescent

pseudomonads which are among the most common and effective biocontrol agents for a

broad-spectrum of pathogens (Weiler 1988).

The first step to 'fixing' biocontrol has been to understand how it works.

Various biocontrol mechanisms have been elucidated using molecular and genetic

6

analysis (Loper et al. 1997, Thomashow and Mavrodi 1997). The primary mechanism in

many Pseudomonas systems is production of antimicrobial compounds such as

pyoluteorin (PLT), 2,4-diacetylphloroglucinol (PHL), and hydrogen cyanide (HCN)

(Keel and Défago 1997, Keel et al. 1992, Maurhofer et al. 1994, Voisard et al. 1989).

Biosynthesis of these compounds is regulated by a two-component system comprising

the response-regulator GacA that is transcriptionally activated by the membrane-bound

sensor-kinasc GacS (formerly ApdA, LemA: Kitten et al. 1998). The importance of

functional gacS-gacA genes in biocontrol of soilborne fungal pathogens has been

confirmed using constructed mutants (Corbell et al. 1995; Gaffney and Lam et al. 1994;

Lavilleetal. 1992).

This work has opened new doors for improving strain activity. Amplification of

regulatory elements and/or biosynthetic loci, and heterologous expression of antibiotic

genes from other strains have been the more popular approaches to improve biocontrol

strains (Haas and Keel 1997). For example, Schnider et al. (1995) cloned the rpoD

house-keeping sigma-factor gene in CHAO. When extra copies of the gene were

reintroduced into CHAO. production of both PHL and PLT was increased 4-8 fold.

Biocontrol of Pythium damping-off on cucumber also was improved. The biocontrol

group of Novartis recently reported that increasing gacA copy-number, improving the

starting codon for gacA, replacing the gacA promoter with a constitutive tac promoter,

and increasing copy-number of pyrrolnitrin biosynthetic genes substantially improved

the ability of P. fluorescens BL915 to suppress Rhizoctonia solani on impatiens and

cucumber (Ligon et al. 1996). Heterologous expression of phcnazine biosynthetic genes

in PHL-producing strains improved biocontrol of wheat root disease (Hara et al. 1994),

and expression of salycilic acid genes from the opportunistic human pathogen P.

aeruginosa in the plant-associated biocontrol-inactive P. fluorescens P3 conferred the

ability to induce systemic resistance in tobacco against mosaic virus (Maurhofer et al.

1998). Wilson et al. (1998) demonstrated that biocontrol activity can be improved by

enhancing compatibility (thus ecological competence) of a biocontrol strain with the

host plant. They accomplished this via heterologous expression in epiphytic

Pseudomonas syringae of catabolic genes for unusual nutritional sources (i.e., amino-

acid derivatives called mannopine), and then applying the biocontrol agent on transgenic

crops designed to exude these opines.

7

Understanding the mechanisms of biocontrol also makes it possible to develop

more effective screening procedures to find better strains. PCR-based detection could be

used to identify strains carrying biosynthetic loci for specific antimicrobial compounds

(Stabb et al. 1994, Thomashow and Mavrodi 1997, Weiler et al. 1997), root-colonizing

or competition factors (Loper et al. 1997. Lugtenberg et al. 1996, Wilson et al. 1998),

and induced-resistance determinants (van Loon et al. 1998). Selection procedures could

further be stream-lined by finding strains that can express these genes in different

environments.

The next step then is to understand what triggers regulation, what environmental

signals activate GacS. Endogenous molecules termed autoinducers (e.g., A-acyl-

homoserine lactones) that are produced by the bacterium itself modulate phenazine

antibiotic biosynthesis in P. aureofaciens (also referred to as P. chlororaphis) strain 30-

84 in the wheat rhizosphere (Pierson et al. 1998) in a density-dependent fashion. In

other words, strain 30-84 produces phenazine only when sufficient autoinducer

accumulates in its immediate environment, and this happens only when there are

sufficient cells of strain 30-84 present producing the signal. However, strain 30-84 can

cross-talk with other bacteria in the rhizosphere community utilizing their autoinducer

signals to initiate antibiotic production, perhaps even when its own population density is

too low (Pierson et al. 1998). It may thus be possible to improve ecological competence

and biocontrol in the future by constructing or isolating strains that can translate

multiple bacterial 'languages', such as Raaijmakers et al. (1995) demonstrated with

transgenic P. fluorescens able to utilize heterologous siderophores.

Much less though is known about exogenous environmental signals influencing

biocontrol. Understanding these signals is key to improving the ability of strains to

produce antimicrobial compounds and to suppress disease in the diverse environments

where they are applied and expected to work. Armed with such information, we could

customize strains for use in particular environments, modify environments to be more

favorable to strains, and/or develop strains that are relieved of environmental signal

control. Minerals would be useful 'environmental signals' for these purposes because:

(i) minerals are central components in over 300 en/ymes and proteins and they influence

diverse biological functions (Berg and Shi 1996, Vallec and Auld 1990); (ii) minerals

are relatively easy and inexpensive to work with in an agronomic setting; and (iii) soil

8

mineral content (and other edaphic parameters) have recently been correlated to the

biocontrol activity of Trichoderma and Pseudomonas. Caution, however must be

exercised when using mineral amendments/nutrients because mineral nutrition may

have the nontarget effect of increasing (or decreasing) the incidence and severity of

various diseases (Engelhard 1989), which may negate any benefit derived from

improved biocontrol activity.

Genetic instability has long been hypothesized to be another potential source of

variability in biocontrol (Weller et al. 1988). Biocontrol would likely be most affected

by mutation in phenotypie traits important for colonization of the infection court and for

mechanisms involved in disease suppression (eg., antibiotic production). Unpublished

data from industry indicate that in large-scale fermentations contamination with as much

as 10% mutants occurs in some Pseudomonas biocontrol strains (S. Hill and N.

Torkewitz, Novartis personal communication). Preliminary data indicate that the gacS-

gacA genes are particularly unstable in biocontrol pseudomonads (Voisard et al. 1994),

just as has been reported for many pathogenic bacteria (Kitten et al. 1998).

The overall objective of this thesis was to identify factors (signals) that influence

the biocontrol activity of diverse P. fluorescens strains. Direct and indirect effects of

minerals and pathogen metabolites on the stability of gacS-gacA regulatory genes

(Chapter 1), bacterial secondary metabolism (Chapter 2), and disease suppression

(Chapter 3) were examined. Potential nontarget effects of zinc and other minerals on

tomato root disease (Chapter 4) and on growth of beneficial bacteria (Chapter 2) were

also investigated.

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Voisard, C, C. T. Bull, C. Keel, J. Laville, M. Maurhofer, U. Schnider, G. Défago, and D.

Haas. 1994. Biocontrol of root diseases by Pseudomonas fluorescens CEIAO: current

conceps and experimental approaches, p. 67-89. In: F. O'Gara, D. N. Dowling, and B.

Boesten (eds.), Molecular Ecology of Rhizosphere Microorganisms: Biotechnology and the

Release of GMO's. VCH, Weinheim, Germany.

Weller, D.M. 1988. Biologcial control of soilborne plant pathogens in the rhizosphere with

bacteria. Annu. Rev. Phytopathol. 26:379-407.

Weiler, D.M., Raaijmakers, J.M., and Thomashow, U.S. 1997. The rhizosphere ecology of

antibiotic-producing pseudomonads and their role in take-all decline, p. 58-64. In: A.

Ogoshi. K. Kobayashi, Y. Homma, F. Kodama. N. Kondo, and S. Akino (eds.), Plant

Growth-Promoting Rhizobacteria - Present Status and Future Prospects, Japan-OECD Paris

workshop, Hokkaido University, Sapporo, Japan.

Wilson, M., Moss, W.P.. Ji, P., Wang. S.-Y., Dianese, A.C., Zhang, D., and Campbell, H.L. in

press. Molecular approaches in the development of biocontrol agents of foliar and floral

bacterial pathogens. In: B.K. Duffy, U. Rosenberger, G. Défago, (eds), Molecular

Approaches in Biological Control, IOBC/WPRS Bulletin.

*^ 4! «

ff %

,- f

k i. S, >ü«H

13

Chapter 1

Mineral Compounds Reduce the Accumulation of Spontaneous Regulatory

Mutants During Liquid Culture of Pseudomonas fluorescens Biocontrol

Strains

Abstract

Secondary metabolism in many fluorescent pseudomonads is regulated by a two component

sensor kinase-response regulator system comprising the gacS and gacA gene products.

Mutation in either gene blocked production of the antimicrobial compounds hydrogen cyanide,

2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitnn by the model biocontrol strain

Pseudomonas fluorescens CHAO. Mutants also had an altered ability to utilize several carbon

sources and to increase medium pH compared with the wild-type, suggesting that gacS and

gacA may influence primary as well as secondary bacterial metabolism. The biocontrol activity

of CHAO inoculants against Pythium damping-off of cucumber was significantly reduced with

10% GacS- or GacA- mutants and was almost absent with 50% or more mutants, demonstrating

the potential risk associated with genetic instability. Spontaneous biocontrol-negative

regulatory mutants accumulated at a high frequency during liquid culture, accounting for more

than 1% of the total viable cells after 12 days. Occurrence of mutants complemented with

clones of gacS and gacA was proportional, indicating similar selective pressures for each

mutant type. Mutants could be easily distinguished from the wild-type based on their orange-

colored, enlarged colony appearance. In a simulation of an industrial inoculant fermentation

process, nutrient rich medium with a high electrolyte concentration favored mutants during

scale-up, giving approximately 7, 23. and 61 % mutants accumulating after transfer to 20, 100,

and 500 ml volumes, respectively. One-tenth media dilution, and media amendments of zinc,

copper, cobalt, manganese, and ammonium molybdate increased competitiveness of the wild-

type and substantially reduced the accumulation of gacS and gacA mutants. Spontaneous and

genetically-engineered mutants had similar responses to cultural conditions. Zinc and media

dilution reduced the accumulation of spontaneous gacA mutants of other P. fluorescens

biocontrol strains from Ghana and Italy. Possible mechanisms for mutant accumulation and

how mineral amendments and media dilution reduce this are discussed.

14

Certain plant-associated bacteria, particularly fluorescent Pseudomonas spp., have been

exploited for suppression of crop diseases, and their importance in agriculture is

expected to escalate (Cook 1993). Commercial development of biocontrol entails the

large-scale production of bacterial inoculants. Typically, bacterial inoculants, regardless

of their intended use (e.g., agricultural, pharmaceutical, food processing,

manufacturing), are mass-produced in industrial fermentors with small batches used to

inoculate increasing fermentation volumes, a process often referred to as scale-up

(Smith 1987). A stream-lined process (i.e., cost effective) that delivers high yield and

optimal efficacy is the primary objective in fermentations designed to recover viable

cells. Culture media are prepared from an eclectic assortment of ingredients and are

generally nutrient rich which does not reflect most natural bacterial environments. This

is particularly evident for biocontrol agents originally isolated from the rhizosphere or

phyllosphere where nutrients are often limiting. Considering this and the scale-up

process necessary to prepare large volumes, liquid fermentation of bacterial inoculants

is disturbingly similar to repeated transferring and prolonged incubation times in

artificial growth media« laboratory practices long known to generate spontaneous

mutation in microorganisms.

Genetic and molecular analysis has demonstrated that production of various

antibiotics and hydrogen cyanide (HCN) is a primary mechanism of biocontrol for many

strains, accounting for as much as 90 % of their disease suppressive activity

(Thomashow and Weiler 1996). As more biocontrol strains arc analyzed, it is becoming

apparant that biosynthesis of these antifungal secondary metabolites in Pseudomonas

spp. is commonly controlled by a two component system comprising the sensor-kinase

GacS and the response-regulator GacA, or functional and molecular homologs (Corbell

and Loper 1995, 1996, Gaffney et al. 1994, Laville et al. 1992, Pierson et al. 1998,

Thomashow and Mavrodi 1997). GacS- and GacA- mutants are typically less inhibitory

to fungal pathogens, presumably due to loss of antibiotics and hydrogen cyanide (HCN)

(Thomashow and Mavrodi 1997). The gene gacS is the new designation for lemA of P.

syringae and homologs apdA, repA. and pheN (Kitten et al. 1998).

Despite obvious potential risks involving instability of gacS and gacA or other

genes important in biocontrol (Schroth et al. 1984. Weiler 1988), little if any effort has

been made to document, to understand, much less to control this problem during

15

inoculant production. Here we report the accumulation of a high frequency of

spontaneous GacS- and GacA- mutants in liquid cultures of the Swiss biocontrol strain

P. fluorescens CHAO. The importance of gacA in biosynthesis of the antifungal

metabolites. 2,4-diacetylphloroglucinol (PHL). pyoluteorin, and HCN, and the role of

this gene in fungal inhibition and biocontrol activity of strain CHAO has previously been

demonstrated using gene-replacement and transposon insertional mutation (Laville et al.

1992). Less is currently known about the function of gacS in this strain (Carruthers and

Haas 1998). Our objectives were first to phenotypically characterize these spontaneous

regulatory mutants and to determine their impact on the biocontrol efficacy of bacterial

inoculants. We then set out to identify the selective pressures that favor mutant

accumulation during inoculant production and to develop a cost-effective approach to

minimize genetic instability in P. fluorescens biocontrol strains. A preliminary report of

this work has been published (Duffy and Défago J 995).

Materials and Methods

Bacterial strains, mutant derivatives, and culture media. Strains and plasmids used

in this study are described in Table 1. Wild-type strain CHAO was originally isolated in

1983 from a Swiss sandy loam naturally suppressive to tobacco black root rot (Stutz el

al. 1986). An archival sample from 1985, kept at -80 °C, was used in this study. Strains

CHA510. CHA89, and CHA96'" are genetically-engineered regulatory mutant

derivatives of CHAO. Spontaneous regulatory mutant derivatives CHAS9, CHAS17,

and CHAS45 were isolated from stationary-phase nutrient broth cultures and mutant

CHASP1 was isolated from tobacco roots that had been inoculated with CHAO wild-

type and grown under gnotobiotic conditions for 6 weeks. Wild-type PGNL1, PGNRL

and PGNR4 were isolated from tobacco roots grown in a Ghana silt loam suppressive to

tomato root diseases; and wild-type PINR2 and PTNR3 were isolated from tomato roots

grown in an Albenga, Italy sandy loam suppressive to Fusarium wilt. Spontaneous

mutants of these strains were isolated from orange sectors that appeared in colonies

grown for 10 to 14 days on King's medium B (KB) agar (King et al. 1954). Plasmids

used for genetic complementation were vectored by Escherichia coli. AU bacteria were

stored in dilute 0.08% nutrient broth (Difco, Detroit. MI) with 40% glycerol at -80 °C.

Fresh cultures were started from glycerol stocks for each experiment by plating onto KB

agar.

16

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristics' Source or

reference

Pseudomonas fluorescens

CHAO

CHA89

CHA96"'

CHA5I0

CHAS9

CHAS45

CHAS17

CHASP1

PGNL1

PGNL1S2

PGNR1

PGNR1S1

PGNR4

PGNR4S2

PINR2

PINR2S3

P1NR3

PINR3S1

Anl 2 HPT \ Flu 2 DSJ,CmRwild-type

Ant ; HPT ". Flu A DS ". Ki/

gene-replacement gacA mutant of CHAO

Ant", HPT '.Flu ADS". Rif.

gacA '-'kicZ genc-rcplaccnicnt gacA mutant

of CHAO

Ant". HPT ".Flu ADS'. KmR

gacS;:T\\5 mutant of CHAO

Ant ". HPT .Flu A DS

'

spontaneous gacA mutant of CHAO

Ant ", HPT ,Flu A DS

"

spontaneous gacA mutant of CHAO

Ant ", HPT ". Flu A DS"

spontaneous gacS mutant of CHAO

Ant", HPT", Flu'*. DS"

spontaneous gacS mutant of CHAO

Ant+. HPT \ Flu \ DS 2 wild-type

Ant", HPT", Flu*'

spontaneous gacA mutant of PGNL1

Ant '. HPT \ Flu +. DS 2 wild-type

Ant ".HPT ".Flu"

spontaneous gacA mutant of PGNRl

Ant ". HPT+, Flu r. DS \ wild-type

Ant.HPT ", Flu

^

spontaneous gacA mutant of PGNR4

Ant '. HPT 2 Flu \ DS 2 wild-type

Ant .HPT .FUG"

spontaneous gacA mutant of PIXR2

Ant 2 HPT 2 Flu 2 DS ', u, Id-type

Ant ". HPT ", Flu+"

spontaneous gacA mutant of P1NR2

Stutzetal. 1986

Laville et al. 1992

Natschetal. 1994

C. Keel G. Defago

C. Voisard, D. Haas

C. Voisard, D. Haas

C. Voisard. D. Haas

This study

Keelctal. 1996

This study

Keel et al. 1996

This study

Keel et al. 1996

This study

Keelctal. 1996

This study

Keel et al. 1996

This study

17

Escherichia coli

DH5a

ED8767

HB101

Plasmids

pME3066

pJEL5771

pME497

endAl hsclRU (rK-mK+) supE44X

thi-1 recAl gyrA96 rclAl phoA dcoR

cr>80d/örZAM15 A(lacZYA-argF)ll 169

metB hsdS supE supF recA56

F hidS20 supE44 recA13 aral4

proA2 lacYl gal'K2 rpsL20 lcu-6 thi-1

x\l~5 mtl-1 (str-20\\{mcrC-mrr)

IncP-1 rcplicon. Mob0. Te\

contains a functional gacA (Y49) gene

from CHAO

TcR, contains a functional gacS (syn.

a/x/A)gcne from P. fluorescens strain

PP5

IncP-1 rcplicon. RcpA(ts). Tra\ Km

Sambrook et al.

1989

Murray 1977

Boyer 1969

Lavilleetal 1992

Corbclletal 1995

Voisard et al 1988

'Ant = antibiotics (2.4-diacetylphloroglucinol and pyolutconn): HPT = hydrogen cyanide, protease,

tryptophan-sidc-chain oxidase; DS = disease suppression; Flu = fluorescent siderophorcs (pyoverdinc and

pyochclin). For these characters, superscripts '+' indicate wild-typc production levels, '++' indicate

overproduction, '-'reduced or absence of production. ApR, Cm', KmR. Tc\ Ril* = resistant to ampicillin,

chloramphenicol, kanamycin. tetracycline, and rifampicin. respectively.

Liquid cultures were grown in normal strength, nutrient broth-yeast extract

(NBY) prepared with 0.8 % nutrient broth and 0.5 % yeast extract (Difco) in twice

distilled H20, pH 6.5. Single lots of nutrient broth and yeast extract were used

throughout this study. Prepared NBY broth had (mg/L): total nitrogen (1441.0), amino

nitrogen (604.0), phosphate (600.1), potassium (597.9), sodium (259.7), chloride

(121.7), sulfate (54.9). magnesium (22.9), calcium (6.1), zinc (0.5), cobalt, copper, iron,

manganese, tin and lead (<0.1 ). Media conductivity, a measure of electrolyte

concentration, was determined using a Volmatic conductivity meter LM20 (Volmatic

SARL, Maze, Switzerland) and pH was determined with a digital meter (ABS, Zürich,

Switzerland).

Mutant characterization. Five hundred-seventv eight spontaneous mutants

with a distinct orange-colored colony phenotype were isolated from 192 NBY broth

cultures of wild-type CHAO that were incubated for 12 days. Mutants were analyzed for

genetic similarity to the wild-type using a method based on the polymerase-chain

reaction with randomly amplified polymorphic DNA markers (PCR-RAPDs). The

18

primer D7 obtained from a series of random oligonucleotides (Operon Technologies,

Almeda, CA) provided consistent and distinct banding patterns with polymorphic

markers specific to strain CHAO (Keel et al. 1996). Bacteria were grown in wells of

microtitre plates containing 50 (il of dilute ( 1/10 strength) KB broth and incubated for

24 h at 27 °C with gentle agitation. Sample preparation, PCR-amplification, and gel

electrophoreses methods were as previously described (Keel et al. 1996).

All 578 mutants were tested at least twice for production of HCN (Keel et al.

1996), extracellular proteases (Sacherer et al. 1994), and tryptophan side-chain oxidase

(TSO), an enzyme important in indole-acetic acid biosynthesis (Oberhänsli et al. 1991),

using standard methods. A random sub-sample of 205 of these mutants were then

screened for genetic complementation with gacS and gacA clones. Mobilization of

recombinant cosmids pJEL5771 and pME3066 from E. coli was accomplished by

triparental matings with the helper plasmicl pME497 (Voisard et al. 1988).

Transconjugants were screened for restoration of HCN. protease, and TSO production

on milk agar (Sacherer et al. 1994). Genetically-engineered derivatives CHA510 and

CHA89 were routinely used as controls for successful complementation of gacS and

gacA mutations, respectively.

Five mutants completely complemented with either gacS or gacA were further

characterized for reversion frequency, cell length, carbon-source utilization, pH change

in NBY broth, antibiotic sensitivity, antibiotic and siderophorc production, in vitro

fungal inhibition, and suppression of cucumber damping-off. Reversion frequency was

estimated as the fraction of CFU from 24 h NBY broth cultures of spontaneous mutants

that were protease positive on milk agar. Cell length was determined after 24 h growth

in 20 ml NBY broth by mounting cells onto polycarbonate filters, staining with CHAO

specific antisera and fluorescent antibodies, and measuring the length of 100 cells per

isolate using a Zeiss Axioskop cpifluorescence microscope, as previously described

(Troxler et al. 1997). Carbon-source utilization profiles for the wild-type was

determined using the Biolog® GN and GP Microplate141 system according to

manufacturer instructions (Biolog Inc., Hayward. CA). Change in pH was determined in

NBY broth after 24 h growth. Tolerance to synthetic PHL (200 to 1000 |ig/ml) and PLT

(50 to 500 (ig/ml) was determined in NBY broth following Keel et al. (1992). High¬

performance liquid chromatography was used to quantify production of pyochelin,

19

salicylic acid, pyoluteorin, and pyrrolnitrin in NBY broth after 48 h incubation; and

production of PHL in NBY broth amended with 1% glucose, as previously described

(Duffy and Défago 1997). The ability of mutants to inhibit Pythium ultimum growth was

determined on KB agar with and without 100 |iM FeClv by spotting 5 (il of overnight

NBY broth cultures at two opposite sides of the plate 5 mm from the edge. After 24 h

incubation at 27 °C, fluorescence around bacterial colonies was observed with a UV

lamp. Then plates were inoculated with P. ultimum by inverting a 4-mm-diameter agar

plug of a 3-day-old culture in the center. The distance between the edge of the bacterial

and fungal colonies (inhibition zone) was measured after 36 h.

Suppression of cucumber damping-off caused by P. ultimum was evaluated in

Eschikon sandy loam (Natsch et al. 1994). Soil was sieved (2.0 mm mesh), infested with

0.5% crushed millet seed colonized by P. ultimum (< 1.0 mm particle diameter), and

incubated 24 h at 20 °C prior to distributing into plastic pots (7.5 cm diameter x 5.5 cm

deep). Bacteria were grown 24 h in NBY broth and collected with centrifugation.

Suspensions of approximately 10" CFU/ml were prepared with 0.5% medium viscosity

sodium carboxymethylcellulose (Fluka, Buchs. Switzerland). Pregerminated (2 days at

24 °C on 0.85% water agar) surface-disinfested cucumber seeds (Cucumis sativus

'Chinesische Schlange') were submerged in bacterial suspensions for 5 min. and planted

0.5 cm deep in infested soil with 10 to 15 seeds per pot. Plants were grown in a climate

chamber at 22 °C with 70% relative humidity and a 16 h photoperiod. Percent seedlings

emerged and standing was determined after 10 days.

Influence of mutant contamination on inoculant efficacy. Bacterial

suspensions of wild-type CHAO. gacS mutant CHAS17, and gacA mutant CIIAS33

were prepared from NBY broth cultures, as above. Suspensions were combined to give a

range of mutant concentrations from 0 to 100%). Pregerminated cucumber seeds were

soaked in the suspensions and planted into Pythium infested soil. Percent seedlings

emerged and standing was determined after 10 days. Treatments consisted of three

replicate pots with 15 seeds each and the experiment was repeated once. Non-bacterized

seeds served as a disease control not included in the analysis.

Four assays to determine the influence of mineral amendments on mutant

accumulation. Unless otherwise indicated, bacteria were grown in 20 ml NBY broth in

100 ml Erlenmeyer flasks and incubated 48 h at 27 °C with shaking at 140 rpm in

20

darkness. Filter-sterilized mineral solutions were added to autoclavcd media to give 1.0

mM [B(OIL), CaCl2-2 H20, FeS04-7 H20, LiCl, MgSOr7 H20, Mo7(NH4)602,4 H20,

MnCl24 H20, NaCl], 0.7 mM (CuS04, ZnS04-7 H20) or 0.1 mM (CoCl,-6 H20).

Cultures were inoculated with 10 jil of 1/10 diluted overnight precultures to give

approximately 10'to 10' CFU/ml. Wild-type precultures had no detectable mutanls (< 1

x 10 7ml). Mixtures of wild-type and mutants were prepared by combining precultures

which were then used to inoculate cultures. Sampling was done by plating appropriate

serial dilutions onto KB agar amended with 30 |ig/ml chloramphenicol (KB""), a natural

antibiotic resistance marker for strain CHAO (Voisard et al. 1988). Other P. fluorescens

strains were plated onto non-amended KB agar. Colonics were enumerated and the

percent orange mutants relative to non-pigmented wild-type colonies was determined

after 5 days.

In the first experiment, the effect of media on accumulation of mutants from a

wild-type culture was determined. CHAO was grown 12 clays in broth cultures of NBY,

NBY plus 0.7 mM CuS04, dilute NBY (1/10 strength), and dilute NBY adjusted to a

conductivity of 4.0 mMhos/m Siemens with 30 mM NaCl, the approximate conductivity

of normal strength NBY broth cultures after 48 h bacterial growth. Cultures were

incubated 12 days and serial dilutions were plated on KB"11 agar. Total CFU and percent

orange mutants were determined from 500 to 3000 colonies per treatment. As a second

measure of mutant accumulation, 94 random colonies from each treatment were tested

for HCN, proteases, and TSO production. Each treatment was replicated ten times, with

two samples taken for each replicate, and the experiment was conducted four times.

The second experiment was designed to mimic industrial fermentation processes

which typically use step-wise scale-up in batch size. Samples (10 (il) taken from 12 day

NBY broth cultures above, with a moderate level of mutants (approximately 1.3 %),

were used to seed 20 ml fresh broths of NBY. dilute NBY. or NBY plus CuSO,. After

48 h with shaking at approximately 110 rpm, total CFU and percent mutants was

determined and 100 pi of these were used to inoculated 100 ml fresh media in 500 ml

Erlenmeyer flasks. These cultures were in turn used to inoculate 500 ml volumes in I

liter flasks. Treatments consisted of three to six replicates, each started from an

independent seed culture, and the experiment was conducted three times.

21

The third experiment examined the influence of a wider range of minerals on the

further accumulation of orange mutants from an initially low but detectable level

(approximately 0.3 %). Bacteria were grown in 20 ml broths of NBY, dilute (1/10

strength) NBY, dilute NBY plus NaCl, and NBY plus one of 11 minerals. After 48 h,

total CFU and the percent mutants was determined. Each treatment consisted of four

replicate broths, and the experiment was conducted four times.

The fourth experiment examined the influence of minerals on competition

between coinoculated wild-type and characterized gacS (CHAS17, CHASP1) and gacA

(CHAS9, CHAS45) mutants. Test cultures were inoculated with a mixture of 80% wild-

type and 20% mutant. After 48 h, total CFU and percent mutants was determined. The

experiment was arranged as a 5 x 14 factorial in a split-plot design with a mainplot of

wild-type and mutant combination and a subplot of culture medium. Because of the

large number of treatments, the experiment consisted of eight replications over time. An

extension of this fourth experiment was designed to determine the relationship between

zinc concentration and mutant accumulation. Here we used characterized spontaneous

mutants (CHAS17 and CHAS45) and compared them with genetically-engineered

mutants (CHA510 and CHA96"1). Mixtures of 90% wild-type and 10% mutants were

used to inoculate NBY broth amended with a range of ZnSO,-7 ILO concentrations (0 to

1.1 mM). Percent mutants was determined after 48 h by plating onto KBtm agar. Percent

CHA96"1 was also determined by plating onto KBLm agar plus 100 fig/ml rifampicin. The

experiment consisted of six replications over time.

Effect of zinc and media dilution on mutant accumulation in other

biocontrol strains. For each strain, mixtures of 99% wild-type and 1% gacA mutant

were used to inoculate broths of NBY, dilute 1/10 strength NBY, and NBY plus 0.7 mM

ZnSOt-7 H20. Mutants of each strain were HCN and protease negative and had an

orange-colored colony phenotype identical to CHAO mutants which was used to

determine percent mutants after 48 h. Treatments were arranged as a 5 x 3 factorial with

a main-plot of strain and a sub-plot of media. Treatments consisted of three replicates

and the experiment was conducted twice.

The influence of minerals and media dilution on growth of wild-type CHAO

and mutants. Wild-type CHAO and spontaneous mutants were grown individually in

broths of NBY, dilute NBY, dilute NBY plus NaCl, and NBY plus minerals. After 48 h,

22

CFU/ml were determined by plating onto KBcm agar. Treatments were arranged in a 6 x

J 4 factorial in a split-plot design with a mainplot of bacterial strain and subplot of media

treatment. The experiment consisted of six replications over time. Growth rates for

CHAO, CHAS17, and CHAS33 were determined by recording OD6no over a period of 0

to 48 h in 150 ml broths of NBY, dilute 1/10 strength NBY, dilute NBY plus 30 mM

NaCl, NBY plus 0.7 mM CuSO, or ZnS04- 7 H:0. Treatments consisted of two replicate

broths.

The influence of culture filtrates on growth of wild-type CHAO and

mutants. Wild-type CHAO and spontaneous mutants were grown individually in 20 ml

broths of NBY or NBY buffered to pH 6.5 with 0.2 M NakLPO, and Na2HP04l2H20.

After 18 h at 27 °C, cultures were centrifuged for 30 min. at 2,790 x g and supernatants

were passed through a 0.2- tim-pore-size filter with a borsilcate prefilter (Nalgenc,

Rochester, New York). Culture filtrates were then inoculated with either the wild-type

or mutant. After a further 18 h incubation, CFLT were determined. Treatments were

arranged in four mini-blocks each with the wild-type and one spontaneous mutant

(CHAS9, CHAS45, CFIAS17. or CHASP1). Each treatment consisted of three replicate

broths and the experiment was repeated once. Data for each miniblock were analyzed

separately.

Data analysis. Bacterial CFU data were transformed using the logarithmic base

10 and percentage data were transformed using the arcsine of square roots prior to

analysis of variance. Unless indicated otherwise, treatments were arranged in a

randomized complete block design and experiments were repeated two to four times.

Data from repeated trials were pooled after confirming in preliminary analysis that the

trial x main effects interaction was not significant and/or that variances between trials

were homogenous according to an F-test or Bartlett's test (Gomez and Gomez 1984).

For most experiments, main effects and interactions were further analyzed for

significance with the SAS general linear models procedure (Statistical Analysis Systems

Institute, Gary, NC) with mean comparisons performed using Fisher2s protected least

significant difference (P=0.05) test. SAS regression procedures were used to determine

relationships between mutant content and inoculum efficacy, and between zinc

concentration and mutant accumulation.

23

Results

Mutant characterization and impact on biocontrol. Spontaneous mutants appeared at

a high frequency (approximately 1 %) in stationary-phase cultures of CHAO. Mutants

were easily distinguished from the wild-type in dilution plated samples based on an

unusual colony appearance (i.e., orange color, flattened, expanded, often transluscent,

surrounded by a more intense diffusible, yellow, fluorescent pigment) which increased

in intensity over a period of 5 days. The correlation between this orange colony color

and loss of HCN, protease, and TSO production was approximately 98%. Orange

mutants were indistinguishable from the wild-type in PCR-RAPDs analysis. From 205

orange mutants. 49.7 % were restored to a wild-type phenotype with gacS and 48.2 %

were restored with gacA clones. Of the remaining 2.1 % pleiotropic mutants not restored

with either of these single clones, none were found that required both clones for

complementation. Generally, GacS- and GacA- mutants behaved similarly in all tests

throughout this study, and spontaneous mutants were indistinguishable from

genetically-engineered derivatives.

Spontaneous mutants showed no signs of reversion to HCN, protease, or TSO

positive (< 10'

revertants per ml) after three 48 h subculturings in NBY broth.

Compared to the wild-type, GacS- and GacA- mutants had a clearly reduced and

delayed production of HCN, protease and TSO, and produced no detectable antibiotics.

As in previous studies, mutants were reported simply as negative for these (Table 2).

However, leaky metabolite production was occasionally observed for both spontaneous

and genetically-engineered mutants, particularly with long incubation periods (e.g., > 48

h instead of 24 h for PICN determination). Mutants produced significantly more

pyochelin and salicylic acid, had a significantly larger cell size, and raised the pH of

NBY broth (normally pH 6.5) significantly higher than the wild-type (Table 2). From

128 carbon-sources tested, differences were observed in the ability of spontaneous

mutants to utilize alaninamide. D-malic acid, mono-methyl succinate (increased) and

D,L-a-glycerol phosphate. glycyl-L-glutamic acid, glycyl-L-aspartic acid (decreased)

relative to the wild-type. No differences were observed in tolerance to PHL and PLT

compared to the wild-type.

24

TABLE

2.Phenotypie

characterizationofPseudomonasfluorescensCHAO

spontaneousregulatory

mutants.'

Characteristic

wild-type

GacA-

GacS-

Colonymorphology

onKB

andNBY

Celllength

(um)

Pyoc

heli

n(n

g/10

8CFU)

Salicylicacid(n

g/10

*CFU)

Extracellularprotease

Tryptophan

side-chainoxidase

Hydrogencyanide

2,4-

Diac

etylpholoroglucinol

(ng/

10"CFU)

Pyrr

olni

trin

(ng/

101CFU)

Pyol

uteo

rint

ng/l

O'CF

UjNutrientbrothpH

after48

h

InhibitionzoneofPythiumultimumgrowth(mm)

onKB

onKB

plus

100uM

FeCl

,Cucumber

seedling

standafter7days

inP.ultimum

infestedsoil

(V<)

Circ

ular

,smooth,convex

opaque,beige

3.3

(0.5)

19.2(4.6)

0.6(0.3)

+ + +

61.8(2.5)

2.5

(0.9)

12.5(1.5)

7.77

(0.0

2)

1.03(0.12)

1.43(0

.09)

82.5(3

.8)

Generally

flat,tr

ansl

ucen

t,orange

10

to50%

greaterdiameter

5.8(1.1)

5.3(0.8)

125.5(19.1)

143.9(11.3)

4.2(1.1)

7.1(2.9)

<0.07

<0.07

<0.1

<0.1

<0.07

<0.07

8.04(0

.03)

8.09

(0.0

4)

0.97

(0.0

9)0.93

(0.03)

0.03

(0.0

3)0.07

(0.0

7)

35.7

(5.4

)31.1

(4.2

)

'

Values(±SE)

representdata

forwi

ld-lypeCHAO

and

fivetotenmutantscomplimentedwithgacSandgacA

clones.Eachtreatmentwas

replicated

two

tofivetimes.KB=

King

'smediumB

agar;NBY=nutrient

broth-yeastextractagar.

"+'=strongpositive,

"-'=strongne

gative

reac

tion

,although

some

leakinesswasobservedinmutants.

25

The GacS- and GacA- mutants reduced Pythium growth on KB agar to a similar

level as the wild-type (Table 2). On KB agar, mutants over-produced diffusible

fluorescent pigment typical of pyoverdinc siderophorcs. When the medium was

amended with iron to repress pigment production, the ability of mutants to suppress

fungal growth was abolished. In contrast, the suppressiveness of the wild-type was

actually increased by iron (Table 2), most likely due to antibiotic production. Seed

treatment with mutants was significantly less effective for controlling cucumber

damping-off compared with wild-type seed treatment (Table 2). In fact, the disease

suppressive activity of CHAO inoculants was significantly (P = 0.0001 ; r2 = 0.82)

reduced by increasing concentrations of GacS- and GacA- mutant contaminants, and

was essentially lost with over 50% contamination (Fig. 1).

too

T3c 80CO

Zn

O)

E 60

TSCD

CD

Z 40c

CDO

I 20

0-'

0 20 40 60 80 100

Percent mutants in inoculum

Figure 1. Influence of mutant contamination on biocontrol efficacy of CHAO inoculants.

Cucumber seeds were treated with suspensions of wild-type CHAO inoculum contaminated by

adding GacS- (CHAS17) or GacA- (CHAS33) mutants at a range of concentrations from 0 to

100%. Seeds were grown in soil infested with Pythium ultimum. Percent seedlings emerged and

standing after 10 days. Values represent means of six replicates (± SE). A non-treated control

received no bacteria and had a percent seedling stand of 5%.

Reduction of mutant accumulation with mineral amendments and media

dilution. Four approaches were taken to evaluate the influence of culture conditions on

the accumulation of regulatory mutants in NBY broth. First, we determined the

influence of copper amendment, media dilution, and electrolyte concentration on the

k CHAS17

D CHAS33

No bacteria control

26

appearance of mutants in wild-type cultures that had no detectable mutants at the start.

All treatments significantly reduced the accumulation of orange mutants compared to

the normal strength NBY broth, with 1/10 dilution of NBY providing the best control of

mutant accumulation (Table 3). The validity of using the orange colony color to identify

mutants was supported by the fact that nearly identical results were obtained when

randomly sampled colonies from each treatment were tested for HCN, protease, and

TSO production. In NBY broth, approximately 1% of the colonies were negative for

these metabolites. In comparison, no negatives were observed in dilute NBY, and only

0.2 and 0.3 % were negative in copper-amended and dilute NBY plus NaCl,

respectively. Total bacterial growth was reduced in all treatments compared with normal

NBY broth (Table 3).

Table 3. Mutant accumulation in wild-type CHAO after 12 days in nutrient broth yeast extract

medium (NBY) with copper or dilution'.

Total bacteria

Media (logH)CFU/m1) Percent orange mutants

NBY 9.16 1.19

NBY plus 0.7 mMCuSO, 9.05 0.52

1/10 Dilute NBY 8.99 0.02

1/10 Dilute NBY plus 30 mM NaCl 8.92 0.25

F-LSD00S 0.04 0.45

'Broths were inoculated with overnight CHAO cultures that had no detectable mutants. Total

viable bacteria and percent mutants with an orange colony color were determined after 12 days.Values represent the mean of 40 replicate cultures from four trials (no significant treatment trial

x interaction). Each mam effect was significant (P * 0.0001). Means were compared using

Fisher's protected LSD test.

Second, we examined the problems that might be expected in large-scale

fermentations which typically use small batches to inoculate increasingly larger volumes

of media. When a medium with a selective pressure for mutants, i.e., NBY, was used in

scale-up from 20 to 100 to 500 ml volumes, an exponential increase in mutants was

observed (Fig. 2). In contrast, when a medium that favored the wild-type over mutants

was continually used, i.e., dilute NBY or copper-amended NBY, mutant accumulation

was arrested at all stages of scale-up. Switching from NBY to dilute or copper amended

media at any stage, not only stopped further mutant accumulation, it essentially restored

27

the culture to predominantly wild-type (Fig. 2). Switching clean cultures (i.e., dilute or

copper-amended) to full strength NBY, even for just one cycle, had the opposite effect

of polluting them with mutants.

Seed

1 3

(0 7)__

j ~~~~"~~"~-—--_ '———.

I ~~~~~-—^

~~—~»'

~~——_,

Scale 1

7 1

(15)

A03

(0 2)08

(0 3)

Scale 2

A ^Af

A^ !XA

23 1 08 1 8 01 96 1 0 20 1

(3 1) (0 2) (0 5) (0 1) (18) (0 3) (3 6)

Scale 3

A A "A A^ À \61 5 09 3 4 05 1">0 2 3 30 0

(8 3) (0 3) (10) (0 2) (4 8) (0 8) (6 6)

Figure 2. Mutant accumulation from contaminated seed cultures through three stages of scale-

up from 20 to 100 to 500 ml volumes (Scale 1 to 3). Inoculation and sampling are described in

Materials and Methods. Lines indicate origins of inoculum. Cultures were grown 48 h in

normal NBY (black), dilute 1/10 strength NBY (clear), and NBY plus 0.7 mM CuSO, (grey).

Values below each symbol represent average percent mutants (± SE) for 14 replicate broths.

Building upon these promising results with copper, we then screened a larger

range of minerals. When cultures contaminated with approximately 0.3% orange

mutants were used to inoculate dilute media and NBY broth amended with one of

eleven minerals, we observed a dramatic reduction in mutant accumulation from 25%

in the NBY control to approximately 5% in dilute (1/10 strength) NBY and NBY

amended with copper, zinc, or cobalt (P = 0.0001: Fig. 3). Ammonium-molybdate and

manganese reduced accumulation to approximately 10%. Lithium, iron, boron, and

magnesium slightly reduced accumulation and sodium and calcium had no effect

compared to the NBY control. The beneficial effect of diluting NBY broth was slightly

but significantly diminished by raising the electrolyte concentration with NaCl (Fig. 3).

A similar effect of NaCl added to dilute NBY was observed in another set of

experiments when cultures were inoculated with 10% GacS- and GacA- characterized

mutants (P = 0.0001 ; Fig. 4). Zinc, copper, and cobalt were consistently the most

28

effective treatments; and the reduction in mutant accumulation obtained by diluting

NBY broth was always lost with addition of NaCl. There was a significant inverse

relationship between mutant accumulation and zinc-sulphate concentration (P = 0.0001;

xx = 0.88; Fig. 5). Accumulation was cut in half at concentrations of 0.5 mM and almost

completely controlled at concentrations > 1.0 mM, regardless of whether mutants had

defects in gacS or gacA (Fig. 5A and C), or were spontaneous or genetically-engineered

(Fig. 5B and D). For CHA96"1, plating onto rifampicin-amended KBcm agar or using

orange colony color as a marker for determining mutant accumulation gave nearly

identical results, further validating our mutant detection method (data not shown). Total

bacterial CFU after 48 h was approximately log 9.4/ml in nonamended NBY and was

essentially unchanged by zinc-sulphate concentrations < 0.8 mM. Increasing toxicity

was observed at concentrations of 1.0 and 1.5 mM with average growth reductions of

0.2 and 0.9 log units, respectively (data not shown).

Culture media treatment

Figure 3. Effect of minerals on competition between CHAO and spontaneous orange mutants.

Broths of NBY (control), dilute 1/10 strength NBY, dilute NBY plus NaCl, or NBY plus

minerals were inoculated with a low but detectable level of orange mutants (approximately

0.3%). After 48 h, percent mutants was determined. Bars represent the mean of 16 cultures (+

SE). Fisher's protected LSD value = 6.71 percent.

29

c

3 20-

C 100-

80 -

liLillllllllU BO-

°

IlL.llIllM

HU .llllllllO Q + o N

Figure 4. Effect of minerals on competition

between CHAO and characterized GacA- (A,

CHAS9; B, CHAS45) and GacS- (C,

CHASP1 ; D, CHAS17) mutants. Broths (see

Fig. 3 legend) were inoculated with a

bacterial mixture containing 90% wild-type

CHAO and 10% mutant. After 48 h, percent

mutants was determined. Bars represent the

mean of eight cultures (+ SE). Fisher's

protected LSD values = A, 10.4; B, 8.2; C,

11.3; D, 6.8 percent.

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

ZnS04*7H20 (mM)

Figure 5. Relationship between zinc concentration and mutant accumulation. Broths of NBY

amended with a range of zinc concentrations were inoculated with a mixture containing 90%

wild-type CHAO and 10% spontaneous (A, CHAS17: C, CHAS45) and genetically-engineered

(B, CHA510; D, CHA96"') mutants. After 48 h. percent mutants was determined. Values

represent the mean of six cultures (± SE). Regression lines approximate 6 \ - 26 x + 25 (P <

0.0001; r?> 0.93).

30

Mutation in other biocontrol strains. Spontaneous mutants defective for HCN,

protease, and the antibiotics PHL and pyoluteorin were readily recovered from five

wild-type biocontrol pseudomonads isolated from tobacco roots grown in soil from

Ghana (PGNR1, PGNR4, PGNL1) and Italy (PINR2, PINR3). Orange colored,

translucent sectors composed of regulatory mutants formed in colonies grown for an

extended period (about 10 days) on KB agar. Over-time, mutants eventually over-grew

the wild-type. These orange mutants were phenotypieally identical to those observed

with strain CHAO. When the wild-types were paired with 10 % GacA- mutants, mutant

accumulation was reduced with zinc amendment and media dilution (P = 0.0289)

compared with full strength NBY broth (Table 4). This was true for all five strains.

However, a significant strain x media interaction (P = 0.0113) indicated that some

strains benefitted more than others from zinc and media dilution. In zinc-amended NBY,

reductions in mutant accumulation relative to non-amended NBY ranged from 4.6-fold

Table 4. Influence of zinc amendment and media dilution on accumulation of spontaneousGacA- regulatory inutants of biocontrol strains from Ghana and Italy .

Percent mutants'

NBY

Origin Strain NBY plus zinc Dilute NBY F-LSD0(h

Ghana

PGNL1 31.7 2.3 1.6 8.0

PGNR1 16.8 2.6 2.7 8.6

PGNR4 22.3 4.8 23 6.8

Albenga, ItalyPINR2 29.2 1.7 3.0 3.7

PINR3 40.4 4.0 9.3 14.2

F-LSD,, 14.7 1.4 o ">

y Nutrient broth-yeast extract (NBY). NBY with 0.7 mM ZnSO, • 7 H,0 (zinc), or with 1/10

dilution (dilute NBY) were inoculated with 992c wild-type and 1% GacA- mutant. Percent

mutants was determined after 48 h growth as described in Materials and Methods.

'

The strain x media interaction was significant (P = 0.01 13) and data were analyzed based on

response to individual mam effects. Values represent the means of six replicate broths.

Differences observed within a row or column using Fisher's protected LSD are significant at (P

< 0.0289).

31

for PGNR4 to 17.2-fold for PINR2. In diluted NBY, reductions ranged from 4.3-fold for

PINR3 to 19.8-fold for PGNL1, relative to non-amended NBY. It was also evident that

some strains (i.e. PINR3) were more susceptible to mutant accumulation than others

regardless of media (Table 4).

Relationship between mineral effects on growth and mutant accumulation.

A significant mineral x strain interaction, and significant main effects (P = 0.0001)

indicated that media treatments had a differential influence on the yield of culturable

bacteria after 48 h growth. Generally, for any given medium there were no consistent

differences between the wild-type and mutants (Table 5. vertical comparisons), and no

apparent relationship between media effects on growth and mutant accumulation (Fig.

4). For example, in zinc amended NBY growth of one GacA- mutant, CHAS9, was

slightly reduced compared with non-amended NBY but growth of another GacA-

mutant. CHAS45. was not affected (Table 5). In copper amended NBY, the reverse was

true and growth of CHAS45 but not CHAS9 was reduced. Zinc and copper did not

affect growth of either of the GacS- mutants, nor of the wild-type. However, both zinc

and copper reduced accumulation of all mutants in competition experiments (Fig. 4).

Furthermore, cobalt, which also reduced mutant accumulation (Fig. 4), did not affect

growth of any of the mutants but reduced growth of the wild-type (Table 5). Growth of

all strains was reduced in dilute NBY and dilute NBY plus NaCl, but there were

generally no differences among strains (Table 5. horizontal comparisons).

Effect of culture-filtrates on growth of CHAO and mutants. Media changes

resulting from growth of the wild-type or mutants had differential effects on subsequent

bacterial growth (P = 0.02 II; Fig. 6). Generally, culture-filtrates of the wild-type

stimulated growth of both the wild-type and mutants (Fig. 6). This effect was pH

independent, being observed in both non-buffered (Fig. 6A) and buffered NBY broth

(pH 6.5; Fig. 6B). Conversely, filtrates of inutants supported lower bacterial growth, and

pH did have an effect m this case. In non-buffered media, growth of the mutants and the

wild-type were reduced to similar levels (Fig. 6A). However, in buffered media, growth

of the mutants tended to be significantly lower than the wild-type (Fig. 6B).

32

TABLE

5.Effectofli

quid

mediaonbacterialgrowth.y

logCFU/ml

Mediatreatment

Conductivity

NBY

2.4

CuSO,

2.4

ZnSO,

2.3

CoCl,

2.4

Mo,(NH,2A

2.9

MnCl,

2.3

LiCI

2.4

FeSO,

2.3

BUG

2.2

MgSO,

2.4

NaCl

2.3

CaCi,

2.4

1/10DiluteNBY

0.2

1/10DiluteNBY

pus30mM

NaCl

4.0

CHAO

CHAS17

CHASP1

CHAS9

CHAS45

F-LSD

F-LSD.

9.67

9.66

9.61

9.37

9.59

9.68

9.61

9.54

9.65

9.67

9.67

9.67

8.86

8.93

0.10

9.48

9.52

9.58

9.46

9.55

9.49

9.53

9.50

9.58

9.53

9.48

9.56

8.86

8.89

0.14

9.57

9.54

9.69

9.36

9.67

9.63

9.61

9.59

9.63

9.68

9.59

9.65

8.94

8.98

0.13

9.39

9.55

9.28

9.00

9.19

9.60

9.38

9.45

9.44

9.66

9.40

9.57

9.39

9.51

9.38

9.57

9.44

9.58

9.51

9.48

9.35

9.57

9.51

9.53

8.82

8.89

8.85

9.01

0.15

0.16

0.20

ns

0.11

0.13

0.16

0.12

0.13

0.14

0.18

0.11

0.07

0.08

0.16

0.14

5

Wild

-typ

eCHAO,gacS(CHAS17,CHASP1),andgacA(CHAS9,CHAS45)

mutantswereinoculatedin

divi

dual

lyinto20ml

nutrientbroth-yeast

extract(NBY),NBY

amendedwithminerals,ordilutedto

1/10st

reng

th.Sodium

chloridewasadded

todiluteNBY

toincreasemedium

conductivity

(mMho/mSiemens).Total

viablecellcounts

(log

CFU)

weredetermined

after48

h.'

Valuesrepresentthemeans

ofsixre

plicates.The

interactionofmediax

strainwas

sign

ific

ant(P<0.0001)anddatawereanalyzed

ontheresponseto

media(P<0.0001,withinacolumn)and

strain(P

<0.0429,withinarow,exceptwhere

'ns'

indicatesan

insi

gnif

ican

tANOVA).Meanswere

comparedusingFisher'sprotectedLSD

test.

33

9.4-

E 8.2-1

76-

III Uli lili Ulio

94-

o i-ift

12-1 I

76-k

^ os ï; as ît m :*£ m ^ N- i£ n. 22 t ^ t-

5^g£ S^S g^3s Si^a.ggww gsjij 5§st SStro.

Strain inoculated:culture media source

Figure 6. Bacterial growth m culture-filtrates of wild-type CHAO (WT) or mutants (CHAS9,

CHAS45, CHAS17, CHASPl). A, Filtrates of NBY broth; or B, pH 6.5 buffered NBY broth

were taken after 18 h bacterial growth, filter sterilized, and re-inoculated with bacteria. After a

further 18 h incubation, bacterial growth (log CFU) was determined. Wt:Wt indicates wild-type

CHAO grown in wild-type filtrates; Wt:S9 indicates wild-type CHAO grown in CHAS9

filtrates; S9:Wt indicates CHAS9 grown in wild-t>pe filtrates: and S9:S9 indicates CHAS9

grown in CHAS9 filtrates. Treatment setup and designations are similar for wild-type

cominations with mutants CHAS45, CHAS17. and CHASPl. Bars represent the mean of six

replicates (+SE).

Discussion

Continued expansion of emerging microbial inoculant markets relies on product

quality control. Attention has generally focused on optimizing product shelf-life (i.e.,

cell viability), reducing phytotoxicity, and excluding potentially hazardous microbial

contaminants (Olsen et al. 1996. Sündiger et al. 1996. Smith 1987). Our study

documents for the first time that the quality of bacterial inoculants can also be

jeopardized by genetic instability during liquid fermentation. Using the model

biocontrol strain P. fluorescens CHAO. we found that spontaneous mutants with inactive

gacS and gacA genes accumulated at a high frequency in broth culture. Scaling-up

inoculum production into increasingly larger volumes resulted in exponential increases

in mutants until these dominated the cultures, accounting for over 61 % of the total

CFU. We identified a negative relationship between the level of mutant contamination

34

and the biocontrol efficacy of inoculants. Contamination of inoculants with as little as

10 % mutants significantly reduced suppression of Pythium damping-off of cucumber,

while 50 % or more inutants rendered inoculants essentially inactive. Reversion to a

wild-type phenotype occurs at a low frequency (< 1 in 10' cells) if at all (Grewal et al.

1995), and would not be of any practical consequence after application. Reduction of

biocontrol activity was probably due to the inability of mutants to synthesize hydrogen

cyanide, pyoluteorin and 2,4-diacetylphloroglucinol, and other antimicrobial

compounds regulated by gacS and gacA genes. Indeed when spontaneous mutants were

tested alone almost all biocontrol activity was lost, providing further support for

previous studies that used insertion mutants to demonstrate the importance of these

genes and compounds in biocontrol (Thomashow and Weiler 1996).

Dose-response studies have demonstrated that a threshold population density of

bacterial agents is required for significant disease suppression, and that relatively small

population declines can dramatically reduce the level of protection (Raaijmakers et al.

1995, Smith et al. 1997). We extend this idea by specifying that a threshold population

of 'biocontrol-active' cells is needed for effective disease suppression, and that mutant

contamination interferes with inoculant efficacy by lowering the dose of such

biocontrol-active cells. Application of larger doses of contaminated inoculants to

compensate for the lower level of active cells would not only increase production costs

but would likely prove ineffective because mutants appear to be at least as competitive

as the wild-type in plant environments. Mutants would likely preclude the establishment

of a threshold population of effective cells regardless of dose applied and may actually

precipitate a gradual displacement of wild-type cells after application infringing on

biocontrol later in the growing season. We have shown by isolating an GacS- mutant

(CHASPl) from tobacco roots that spontaneous mutants do arise and/or proliferate in

the rhizosphere. Previously. Natsch et al. (1994) found that gacA- insertion mutants of

CHAO were slighlty less competitive in bulk soil, equally competitive in the rhizosphere

and more competitive on the rhizoplane/root interior relative to the wild-type. Similar

results were found for lemA (syn. gacS) insertion mutants of phytopathogenic P.

syringae pv. syringae (Hirano et al. 1997) which displaed reduced colonization of bean

leaves in the field but were equally competitive on germinating bean seeds where loss of

pathogenicity would likely not be ecologically detrimental.

35

Exactly how gacS and gacA modulate bacterial competitiveness in natural

environments is uncertain. Antibiotics regulated by these genes may contribute to the

ecological fitness of biocontrol strains under certain conditions, presumably by

improving competitiveness with sensitive populations of indigenous microbes (Mazzola

et al. 1992). In the biocontrol strain P. aureofaciens 30-84, gacA influences the

expression of other regulatory systems involved in autoinduction and quorum sensing

which in turn influence microbial interactions in the rhizosphere (Pierson et al. 1998).

Recent evidence suggests that overproduction of fluorescent siderophores. as seen when

either gacS or gacA are inactivated, may lead to enhanced endophytic colonization of

plant roots (Duijff et al. 1997). Our results showing altered carbon-source utilization

patterns for mutants, suggest that nutrient competition may also contribute to the fitness

of mutants in certain environments. Furthermore, ApdA- and GacA- mutants had an

altered ability to modify surrounding pH, an indicator of ammonium generation, which

can have a major impact on mineral availability. These results further suggest that gacS

and gacA have a role in primary as well as secondary metabolism.

Spontaneous mutations in gacS and gacA is common among beneficial (Gaffney

et al. 1994, Loper et al. 1997, Thomashow and Mavrodi 1997, Pierson et al. 1998) and

pathogenic pseudomonads (Grewal et al. 1995, Liao et al. 1994, Rich et al. 1994).

Various factors have been implicated as triggers for mutational events and/or selective

pressures for mutant accumulation. We found that the appearance and accumulation of

CHAO regulatory mutants was favored by rich media, which supports similar findings

with P. fluorescens (Loper et al. 1997) and P. syringae (Rich et al. 1994). In contrast,

Grewal et al. (1995) reported that showed that nutrient depletion occuring after

prolonged growth, reduced expression of the pheN locus (syn. gacS), and that this

triggered mutation and genetic rearrangement in the mushroom pathogen, P. tolasii.

Mutations in P. putida (Eberl et al. 1996) Streptomxees (Simonet et al. 1992), and E.

coli (Zambrano and Kolter 1996) have been described as adaptive responses to nutrient

starvation and other stress conditions, particularly as cells enter stationary phase.

Having identified genetic instability as a problem not only in CHAO but in

biocontrol strains from around the world, we then set out to develop approaches to

circumvent it during production of biocontrol inoculants. Mutation could effectively be

controlled by producing inoculants in media with one-tenth dilute nutrient

35

Exactly how gacS and gacA modulate bacterial competitiveness in natural

environments is uncertain. Antibiotics regulated by these genes may contribute to the

ecological fitness of biocontrol strains under certain conditions, presumably by

improving competitiveness with sensitive populations of indigenous microbes (Mazzola

et al. 1992). In the biocontrol strain P. aureofaciens 30-84, gacA influences the

expression of other regulatory systems involved in autoinduction and quorum sensing

which in turn influence microbial interactions in the rhizosphere (Pierson et al. 1998).

Recent evidence suggests that overproduction of fluorescent siderophores, as seen when

either gacS or gacA are inactivated, may lead to enhanced endophytic colonization of

plant roots (Duijff et al. 1997). Our results showing altered carbon-source utilization

patterns for mutants, suggest that nutrient competition may also contribute to the fitness

of mutants in certain environments. Furthermore, ApdA- and GacA- mutants had an

altered ability to modify surrounding pH. an indicator of ammonium generation, which

can have a major impact on mineral availability. These results further suggest that gacS

and gacA have a role in primary as well as secondary metabolism.

Spontaneous mutations in gacS and gacA is common among beneficial (Gaffney

et al. 1994, Loper et al. 1997. Thomashow and Mavrodi 1997, Pierson et al. 1998) and

pathogenic pseudomonads (Grewal et al. 1995, Liao et al. 1994, Rich et al. 1994).

Various factors have been implicated as triggers for mutational events and/or selective

pressures for mutant accumulation. We found that the appearance and accumulation of

CHAO regulatory mutants was favored by rich media, which supports similar findings

with P. fluorescens (Loper et al. 1997) and P. syringae (Rich et al. 1994). In contrast,

Grewal et al. (1995) reported that showed that nutrient depletion occuring after

prolonged growth, reduced expression of the pheN locus (syn. gacS), and that this

triggered mutation and genetic rearrangement in the mushroom pathogen, P. tolasii.

Mutations in P. putida (Eberl et al. 1996) Streptomyces (Simonct et al. 1992). and E.

coli (Zambrano and Kolter 1996) have been described as adaptive responses to nutrient

starvation and other stress conditions, particularly as cells enter stationary phase.

Having identified genetic instability as a problem not only in CHAO but in

biocontrol strains from around the world, we then set out to develop approaches to

circumvent it during production of biocontrol inoculants. Mutation could effectively be

controlled by producing inoculants in media with one-tenth dilute nutrient

36

concentration. Recently, we have found that there is an inverse relationship between

nutrient concentration and mutant accumulation (B. Duffy, unpublished data). However,

dilute media has the disadvantage that cell yield is approximately one log lower. Mineral

amendments were tested in normal strength media. At concentrations that did not affect

cell yield, zinc, copper, and manganese were as effective as nutrient dilution for

improving genetic stability. Minerals have the extra benefit of stimulating antibiotic

biosynthesis in many biocontrol strains (Duffy and Défago, in press). Moreover, either

dilute media or copper amendment can be used to rehabilitate contaminated cultures.

That is, the exponential increase in mutant accumulation in normal strength media can

be halted and reversed by transfcring the culture to media less selective for mutants.

Literature cited

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and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472.

Carruthers, F., and Haas, D. 1998. Signal transduction and the two-component regulatory

system encoded by the lema/gacA genes in Pseudomonas fluorescens CHAO: implications

for biological control, p. 151. In: B. Duffy, U. Rosenberger, G. Défago (eds), Molecular

approaches in biological control, IOBC vvprs Bull. 21(9).

Cook, R. J. 1993. Making greater use of introduced microorganisms for biological control of

plant pathogens. Annu. Rev. Phytopathol. 31:53-80.

Corbell, N., and J. E. Loper. 1995. A global regulator of secondary metabolite production in

Pseudomonas fluorescens Pf-5. J. Bacteriol. 177:6230-6236.

Corbell, N. A., and Loper, J. E. 1996. apdA and gacA genes influence transcription of

pyoluteorin biosynthetic genes in Pseudomonas fluorescens PF-5. Phytopathology 86:S76.

Duffy, B. K., and G. Défago. 1995. Influence of cultural conditions on spontaneous mutations

in Pseudomonas fluorescens CHAO. Phytopathology 85:1146.

Duffy, B. K., and G. Defago. 1997. Zinc improves biocontrol of Fusarium crown and root rot of

tomato by Pseudomonas fluorescens and represses the production of pathogen metabolites

inhibitory to bacterial antibiotic biosynthesis. Phytopathology 87:1250-1257.

Duijff, B. L, Alabouvette, C, and Lemanceau, P. 1997. p. 379. In A. Ogoshi, K. Kobayashi, Y.

Homma, F. Kodama, N. Kondo, and S. Akino (eds.), Plant growth-promoting rhizobacteria:

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37

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40

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41

Chapter 2

Environmental Factors Modulating Antibiotic and Siderophore Biosynthesis

by Pseudomonasfluorescens Biocontrol Strains

Abstract

Understanding the environmental signals that regulate biosynthesis of antimicrobial compounds

by disease suppressive strains of Pseudomonas fluorescens is an essential step towards

improving the level and reliability of their biocontrol activity. We used liquid culture assays to

identify several minerals and carbon sources which had a differential influence on production

of the antibiotics, 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT) and pyrrolnitrin, and

the siderophores, salicylic acid and pyochelin, by the model strain CHAO which was isolated

from a natural disease suppressive soil in Switzerland. Production of PHL was stimulated by

Zn, NH(Mo, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated

by the same factors as PHL. Production of PLT was stimulated by Zn, Co, and glycerol but was

repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a

mixture of Zn and NHtMo. Pyochelin production was increased by Co, fructose, mannitol, and

glucose. Interestingly, production of its precursor salicylic acid was increased by different

factors, i.e., NH(Mo. glycerol and glucose. Mixture of Zn and NH,Mo with fructose, mannitol

or glycerol further enhanced production of PHL and PLT compared with either the minerals or

the carbon sources used alone, but did not improve siderophore production. Extending

fermentation time from 2 to 5 days increased accumulation of PLT, pyrrolnitrin and pyochelin

but not PHL. When findings with CHAO were extended to an ecologically and genetically

diverse collection of 41 P. fluorescens biocontrol strains, certain signals were strain dependent

while others had a general effect. Stimulation of PHL by Zn and glucose was strain dependent;

white PLT production by all strains that can produce this compound was stimulated by Zn and

transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHAO and

seven other strains tested, but to varying degrees. Production of PLT but not pyrrolnitrin by

CHAO was also reduced by 100 mM phosphate. These results (i) provide insight into the

biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for

intentsive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial

inoculants.

Applied & Environmental Microbiology, in press.

42

There exists unquestionable potential for managing plant diseases incited by soilborne

phytopathogens and increasing crop productivity with application of certain root-

associated microorganisms, particularly fluorescent Pseudomonas spp. (Défago and

Haas 1990, Weiler 1988). Interest in biological control has recently intensified because

of imminent bans on effective chemical controls such as methyl bromide, wide-spread

development of fungicide resistance in pathogens, and a general need for more

sustainable disease control strategies. Unfortunately, seemingly inherent variable

performance of most biocontrol strains between field locations and cropping seasons has

hampered commercial development, and relatively few biological agents are registered

for use in production agriculture (Cook 1993). Much of this variability has been

attributed to differences in physical and chemical properties found in natural

environments where biocontrol agents are applied (Duffy et al. 1997, Thomashow and

Weiler 1996). Understanding which enviromental factors are important and how these

influence disease suppression is widely recognized as a key to improving the level and

reliability of biocontrol.

Considerable progress has been made over the past two decades to elucidate the

mechanisms by which fluorescent pseudomonads suppress disease. In diverse crop-

pathogen systems, genetic analysis and direct isolation has demonstrated that the

primary mechanism of biocontrol is production of antibiotics such as 2,4-

diacetylphloroglucinol (PHL), pyoluteorin (PLT). pyrrolnitrin, and phcnazine-1-

carboxylate (Thomashow and Weiler 1996). Under certain conditions, antibiotics

improve the ecological fitness of these bacteria in the rhizosphere which can further

influence long-term biocontrol efficacy (Mazzola et al. 1992). Siderophores, including

salicylic acid, pyochelin. and pyoverdine, which chelate iron and other metals, also

contribute to disease suppression by conferring a competitive advantage to biocontrol

agents for the limited supply of essential trace minerals in natural habitats (Höfte et al.

1992, Loper and Henkels 1997). Siderophores may indirectly stimulate biosynthesis of

other antimicrobial compounds by increasing the availability of these minerals to the

bacteria. Antibiotics and siderophores may further function as stress factors or signals

triggering induction of local and systemic host resistance (Leeman et al. 1996).

Biosynthesis of antibiotics and other antifungal compounds is regulated by a cascade of

endogenous signals including sensor-kinase and response-regulator proteins encoded by

43

apdA (homolog lemA) and gacA (Corbell and Loper 1995, Gaffney et al. 1994, Laville

et al. 1992), sigma factors encoded by rpoD (Schnider et al. 1995) and rpoS (Sarniguet

et al. 1995), and quorum-sensing autoinducers such as A-acyl-homoscrine lactones

(Pierson et al. 1998).

Determining the exogenous environmental signals that modulate the biosynthetic

regulation of antifungal compounds has been comparatively slow, largely because of the

difficulty detecting metabolite production in the soil and rhizosphere (Thomashow and

Weiler 1996). Numerous reporter systems for gene expression have been described

which ultimately will enhance the sensitivity of detection. Reporter systems in

biocontrol pseudomonads have also been used as a preliminary investigative tool to

examine the influence of iron availability on the expression of pyoverdinc genes (Loper

and Henkels 1997) and the influence of Pythium culture filtrates on the expression of

trehalase genes (Gaballa et al. 1997) and genes thought to be involved in rhizosphere

competence (Fedi et al. 1997).

Liquid culture screening is an attractive alternative approach to identify putative

environmental signals because it requires little knowledge of biosynthetic loci, and

because it is more adaptable to the simultaneous detection of multiple metabolites. This

is an important advantage since many of the most effective biocontrol strains produce

several antimicrobial compounds, the relative importance of which probably depends on

the type of soil, host, and pathogen, the stage of disease development, and other

environmental conditions (Thomashow and Weiler 1996, Voisard et al. 1994). Recent

studies suggest that putative signals identified in vitro using liquid culture screening do

indeed act as important environmental signals in natural habitats. For example, we used

liquid culture screening to identify fusaric acid produced by the phytopathogenic

fungus, Fusarium oxysporum f. sp. radicis-lycopersici as a repressor of antibiotic

production by biocontrol pseudomonads (Duffy and Défago 1997a). It was then possible

to demonstrate that fusaric acid acts as a negative signal in biocontrol of Fusarium

crown and root rot of tomato inhibiting antibiotic production in situ (Duffy and Defago

1997a), and that fusaric acid insensitive strains are more suitable for controlling this

disease (Duffy and Defago 1997b).

In the current study, we screened minerals and carbon sources for stimulation or

repression of biosynthesis of several antibiotics (PHL, PLT, and pyrrolnitrin) and

44

siderophores (pyochelin and salicylic acid) by P. fluorescens. Initially, we tested the

influence of these factors on P. fluorescens CHAO isolated from a Swiss soil naturally

suppressive to black root rot of tobacco caused by Chalara elegans (synanamorph

Thielaviopsis basicola. Voisard et al. 1994). P. fluorescens CHAO is a model biocontrol

strain for which the importance of antimicrobial metabolites in disease suppression has

been demonstrated in several crop-pathogen systems, and for which the genetics of

antibiotic and siderophore biosynthesis has been well characterized (Voisard et al.

1994). We then tested glucose, inorganic phosphate, and zinc, three of the most

influential factors with strain CHAO, for influences on antibiotic production by an

ecologically and genetically diverse collection of P. fluorescens biocontrol strains (Keel

et al. 1996). We focused on minerals and carbon sources because (i) they have long been

known to influence the activity of phytopathogenic microorganisms (Engelhard 1989),

(ii) they contribute to the variability of biocontrol in different soils and on host crops

that differ in root exudate composition (Latour et al. 1996, Thomashow and Weller

1996), and (iii) they have been reported to influence production of other antibiotics in

biocontrol strains (Gutterson 1990. Millier et al. 1995. 1996. Slininger and Jackson

1992, Slininger and Shea-Wilbur 1995). Minerals and carbon sources are also appealing

because they are easy and economical to provide during liquid fermentation of

inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous

and introduced bacteria.

Materials and Methods

Strains and cultural conditions. Pseudomonas fluorescens strains used in this study

were isolated from six crop species grown in soils from Ghana, Ireland, Italy,

Oklahoma, Switzerland, and Washington (Table I), and have been genetically

characterized using amplified ribosomal DNA restriction analysis (ARDRA) and PCR-

based fingerprinting with randomly amplified polymorphic DNA (RAPD) markers

(Keel et al. 1996). Bacteria were stored in 0.8r2 nutrient broth plus 0.5% yeast extract

(NBY) broth (Difco, Detroit, Ml) plus 40'2 glycerol at -80 T. Starter cultures were

grown in 10 ml dilute (1/10 strength) NBY broth in 20 ml screw top vials for 8 to 12 h

at 27 °C with 140 rpm. giving approximately 10* CFLVml. Test cultures of 20 ml NB or

NBY broth in 100 ml Erlenmeyer flasks were inoculated with 10 pi of starter culture.

45

TABLE 1. Origin of Pseudomonas fluorescens strains with ARDRA and RAPD grouping '.

OriginStrain (host, soil source) ARDRA group RAPD group

CHAO Tobacco, Morens. Sw itzerland 1 l

Pfl Tobacco. Morens, Switzerland 1 l

Pf-5 Cotton. Texas. USA 1 2

PF Wheat leaves. Oklahoma. USA 1 2

PGNR1 Tobacco. Ghana 1 1

PGNR2 Tobacco, Ghana 1 1

PGNR3 Tobacco, Ghana 1 1

PGNR4 Tobacco, Ghana 1 1

PGNL1 Tobacco, Ghana 1 1

PINR2 Tobacco, Albcnga. Italy 1 1

P1NR3 Tobacco, Albenga. Italy 1 1

CAAI Cucumber, Morens, Switzerland i

CMl'Al Cucumber, Morens. Switzerland i 3

CAPB2 Cucumber. Morens, Switzerland 2 3

PILH1 Tomato. Albenga, Italy 2 5

PITR2 Wheat. Albenga. Italy 2 5

PTTR3 Wheat, Albenga. Italy 2 5

Ql-87 Wheat. Quincy. Washington, USA 0 4

Q2-87 Wheat, Quincy. Washington. USA 2 4

Q4-87 Wheat. Quincy. Washington, USA 2 4

Q5-87 Wheat. Quincy. Washington. USA 2 4

Q6-87 Wheat. Quincy. Washington. USA 2 4

Q7-87 Wheat, Quincy. Washington. USA 2 4

Q8-87 Wheat, Quincy. Washington. USA > 4

Q9-87 Wheat, Quincy, Washington, USA i 4

Q12-87 Wheat, Quincy. Washington. USA 2 4

Q13-87 Wheat, Quincy, Washington. USA 2 4

Q37-87 Wheat. Quincy. Washington. USA 2 6

Q65-87 Wheat. Quincy, Washington, USA 2 3

Q86-87 Wheal. Quincy, Washington, USA 2 4

Q88-87 Wheat. Quincy, Washington, USA 2 4

Q95-87 Wheat. Quincy, Washington, USA 1 3

Ql 12-87 Wheat. Quincy, Washington, USA 1 3

Ql 28-87 Wheat. Quincy. Washington, USA~) 3

Q139-87 Wheat, Quincy. Washington, USA 2 A

TM1A3 Tomato. Morens. Switzerland 2 3

TMPA4 Tomato. Morens, Switzerland i 3

TM1A5 Tomato. Morens. Switzerland 2 3

TMEA5 Tomato. Morens. Switzerland 2 3

TM1B2 Tomato. Morens, Switzeiland 2->

3

P12 Tobacco, Morens, Switzerland 3 8

F113 Smrarbeet. Ireland A 7

'

Strains were isolated from roots of host plant grown in soil collected from the Ghana, Ireland. Italy,Switzerland, and the USA, and characterized with amplified nbosomal DNA restriction analysis

(ARDRA) and randomly amplified polymorphic DNA analysis (RAPD) (Keel et al. 1996).

Chemical analysis indicated that NBY broth contained (mg/L): total nitrogen (1441.0),

amino nitrogen (604.0), total phosphate (600.1), potassium (597.9), sodium (259.7),

46

chloride (121.7), sulphate (54.9), magnesium (22.9), calcium (6.1), zinc (0.5), and

boron, cobalt, copper, iron, lithium, manganese, molybdenum, zinc (<0.1). Autoclaved

media was amended with filter sterilized mineral solutions to give 1 mM BH,Or CaCh •

2H;0, FeSO, • 7 H20, LiCl, MgSOt • 7 H20, MnCk • 4 H20. Mo7(NH4)602t • 4 H20. or

NaCl. 0.7 mM CuS04 or ZnSO, 7 H,0, or 0.1 mM CoCl,- 6 H20. and with autoclaved

stock solutions of carbon sources to give 19f v:v. Cultures were incubated 48 h at 24 °C

with shaking at 140 rpm in darkness, unless otherwise indicated.

Metabolite extraction and detection. Antibiotics and siderophores were

extracted from the culture supernatant and quantified with high-performance liquid

chromatography (HPLC) as previously described (Duffy and Défago 1997a).

Metabolites were identified by comparison with UV spectra of reference compounds.

Metabolite quantity was estimated from standard curves of reference compounds, and

normalized for the number of culturable cells present, which was estimated by spreading

appropriate dilutions on King2s B medium (KB) agar prior to extraction. Liquid cultures

of 20 ml were acidified to pH 2 with 400 to 700 pi of IN HCl and extracted with 20 ml

of ethyl acetate for 30 min. with vigorous shaking at 150 to 200 rpm. Phase separation

was accelerated with 15 min. centrifugation at 4.500 rpm (2,790 g). The organic phase

was transferred to a round-bottom glass flask, flash evaporated, and the residue was

dissolved in 1 ml HPLC grade methanol. Aliquots of 10 |il were injected into a reverse-

phase column (4 x 100 mm) packed with Nucleosil 120-5-Cls and thermostatically

controlled at 50 °C. Maximum absorbances and approximate retention times for

detection were 270 nm, 11.4 min. for PHL (molecular weight 210); 313 nm, 9.4 min. for

PLT (molecular weight 268); 254 nm. 12.1 min. for pyrrolnitrin (molecular weight

257.1); 300 nm, 8.3 min. for salicylic acid (molecular weight 138); and 254 nm, 10.1

and 10.8 min. for the characteristic twin peaks of pyochelin (molecular weight 325).

Mineral compounds and metabolite production by CHAO. Strain CHAO was

grown 48 h in 20 ml broths of NBY. mineral amended NBY. dilute NBY, and dilute

NBY plus 30 mM NaCl. All treatments were tested alone and with 1% glycerol or 1 %

glucose added. Treatments were arranged as a 14 x 3 factorial in a split-plot design with

a main plot of mineral treatment (none, copper, zinc, cobalt, ammonium-molybdate,

manganese, magnesium, iron, boron, calcium, sodium, lithium, 1/10 dilute NBY, dilute

NBY plus sodium-chloride) and subplot of carbon-source amendment (none, glycerol,

47

glucose). The effect of a range of zinc-sulphate concentrations, from 0 to 1.75 mM, on

PHL and PLT production was evaluated in NBY broth. Metabolite production and

bacterial growth were quantified as described above.

Carbon sources and metabolite production by CHAO. Strain CHAO was

grown in NBY broth and in NBY broth amended with carbon sources at 1% v:v.

Carbon-source amendments were tested alone and with a mineral mixture of 0.5 mM

each ammonium-molybdate and zinc-sulphate. Metabolite extractions were made after

48 and 120 h incubation. Treatments were arranged as a 6 x 2 x 2 factorial with a main

plot of carbon source amendment (none, glucose, glycerol, fructose, sucrose and

mannitol), a subplot of mineral amendment (plus or minus), and a sub-subplot of

incubation time (2 and 5 days).

Effect of zinc, inorganic phosphate, and glucose amendments on growth

and antibiotic production by diverse biocontrol strains of P. fluorescens. A

collection of 42 PHL-producing strains (Keel et al. 1996) was grown in broths of NB,

NB amended with zinc-sulphate, and NB amended with 1% glucose. Yeast extract.

which contributed most of the trace amounts of zinc to NBY, was omitted in these trials

without having any effect on bacterial growth. Zinc-sulphate was added to NB at 0.7

mM for ARDRA I strains and at 0.2 mM for ARDRA 2 and 3 strains (Table 1).

Treatments were arranged as a 3 x 42 factorial with a main plot of media amendment

(none, zinc, glucose) and a subplot of strain. Production of PHL was determined for all

strains, and PLT production was determined for ARDRA 1 strains. In a separate

experiment, CHAO and eight additional strains from this collection were grown 48 h in

NB broth containing 19c glucose (except Fl 13 grown with 19é sucrose) and amended

with 0 and 100 mM inorganic phosphate. Production of PHL was determined with

HPLC. For CHAO, additional treatments of 10 and 200 mM phosphate were included.

Production of PLT and pyrrolnitrin was determined for CHAO grown 5 days in NB plus

1 % glycerol.

Statistical analysis. All experiments were conducted at least twice. Treatments

consisted of three to four replicate cultures. Data from repeated trials were pooled after

confirming homogeneity of variances and/or determining no significant treatment x trial

interaction, except in the experiment to determine the influence of zinc and glucose on

antibiotic production by diverse PHL-producing strains. In this case, the large number

48

of treatments required the experiment to be replicated over time, with three replicates

per treatment. Bacterial growth data were normalized with a log 10 plus 1 transformation

prior to analysis Gomez and Gomez (1988). Metabolite quantity was expressed relative

to bacterial growth (approximately 10s CFU/ml) prior to analysis. Significance of main

effects and interactions was determined using the SAS general linear models procedure

(Statistical Analysis Systems, Gary, NO. When appropriate, mean comparisons were

made using Fisher's protected (P < 0.05) least significant difference test. The

relationship between zinc concentration and antibiotic production was evaluated using

SAS linear regression analysis (Pearson coefficient). Relationships between strain

ARDRA or RAPD groupings and PHL production in response to zinc and glucose were

evaluated using SAS correlation analysis.

Results

Influence of minerals and carbon sources on antibiotic production. In the first

experiment, eleven minerals and media dilution were added to NBY medium alone or in

combination with carbon source amendment of \c7c glucose or glycerol to evaluate their

influence on antibiotic production by CHAO after 2 days growth. Mineral amendment

had a significant influence on PHL with or without glycerol or glucose amendment (P <

0.0006), and on PLT with or without glycerol (P = 0.0001) but not with glucose. When

added to NBY, zinc-sulphate and ammonium-molybdate increased PHL production

(Table 2). In general, glycerol increased PHL production. Combination of zinc, copper,

and ammonium-molybdate with glycerol significantly (P < 0.042) increased PHL

production compared with the minerals or the carbon source alone. None of the other

minerals influenced PHL production in NBY broth plus glycerol. Glucose generally

increased PHL production in all mineral treatments, but there were no significant

interactions with mineral amendments. There was a dramatic increase in PHL

production when glucose was added to dilute NBY broth with or without sodium-

chloride. Production of PLT was significantly increased by zinc-sulphate and cobalt-

chloride in normal NBY broth and in NBY plus glycerol compared with the no mineral

controls (Table 2). In general, glycerol increased PLT production. However, glycerol

reduced PLT production in zinc and cobalt media compared with the minerals used

alone. Glucose repressed production of PLT. Manganese, which had no effect on

49

TABLE

2.Influenceofmineralsandcarbon-sourceamendmenton

antibioticproduction

(ng/108CFU)by

P.fluorescensCHAO.'

2,4-Diacetylphloroglucinol

(PHL)

Pyoluteorin(PLT)

NBY

NBY

NBY

NBY

Culturemedia

NBY

plus

glycerol

plus

glucose

LSD

NBY

plus

glyc

erol

plus

glucose

LSD

NBY

alone

<0.1

2.6

98.4

67.5

12.8

27.0

0.9

10.5

CuSO,

<0.1

469.6

913.9

673.6

1.1

19.1

5.8

14.3

ZnS04

6.0

665.1

79.5

509.7

265.8

114.8

13.3

108.9

CoCl,

<0.1

0.5

773.0

706.8

418.2

60.9

5.4

128.8

Mo,(NHt},0„

9.8

186.0

80.1

79.5

26.4

18.7

7.5

17.4

MnCl,

<().

l6.4

113.4

86.2

<0.l

22.7

4.0

5.6

LiCl

"

0.7

2.1

94.5

73.1

34.4

14.5

1.5

16.6

FeSO,

0.2

8.2

17.7

8.8

40.4

45.7

1.9

15.3

B(OH),

<().

!38.2

80.0

ns

38.1

14.2

1.4

13.2

MgSO,

0.1

1.4

241.0

174.5

10.0

20.4

2.1

7.2

NaCl

<0.1

3.6

102.2

75.1

23.6

20.3

1.5

10.1

CaCI,

<0.1

4.5

59.8

38.6

13.5

10.6

0.9

5.1

DiluteNBY

<0.1

58.6

1,93

9.5

1.373.8

6.2

5.9

7.7

ns

DiluteNBY

withNaCl

<0.1

3.2

1,646.9

1,46

3.6

9.2

53.0

10.0

12.3

LSD=

2.5

311.9

948.2

66.0

36.7

ns

'

Within

arow,

foreachmedium,

antibioticconcentrationwas

sign

ific

antl

ydifferentac

cord

ingtoFisher'sLSD

test(P<0.05

forPHL,and<0.02for

PLT),exceptwhere

"ns'

indicatesanonsignificantANOVA.

Withinacolumn,

antibioticconcentrationwas

sign

ific

antl

ydifferentaccording

tothe

LSD

atthebottom(P<0.0006),

exceptwhere

'ns'

indicatesanonsignificantANOVA.

50

antibiotic production when used alone, slightly relieved glucose repression of PLT (Table

2). Mineral amendments did not significantly reduce bacterial growth compared with then

non-amended control; cell number was reduced approximately one log unit by ten-fold

media dilution (data not shown),

0 0.25 0.5 0,75 1

Zinc-sulphate (mM)

Figure 1. Relationship between zinc-sulphate concentration and production of PHL (D) and PLT

() in nutrient broth by CHAO after 48 h growth at 27 °C. Values represent the mean of six

cultures. Regression lines approximate y=140.4x - 15.6 for PHL (solid line; r2=0.8060) and

y=140.8 + 26.0 for PLT (dashed line: r2=0.6329).

Two follow-up experiments examined the relationship between concentrations of

zinc-sulphate and inorganic phosphate with antibiotic production. Zinc-sulphate

concentration, from 0 to 1.1 mM, had a significant (P = 0.0001) positive linear relationship

with production of both PHL and pyoluteorin (Fig. 1). Antibiotic production, normalized

for the number of cells in each culture, continued to increase at concentrations up to 1.75

mM zinc-sulphate (data not shown). Flowever. while bacterial growth was not significantly

affected at concentrations up to Li mM (approximately log 9 CFU), growth sharply

declined to below log 7 CFLI at higher concentrations. Data for concentrations above 1.1

mM were not included in the final regression analysis.

We then tested the effect of a wider range of carbon sources alone and looked at

interactions between carbon source, a mineral mixture of zinc-sulphate (0.35 mM) and

ammonium-molybdate (0.5 mM), and extended incubation time (2 and 5 days). The

interaction of carbon source x mineral x time was significant for both PFIL (P - 0.0334)

51

and PLT (P = 0.0089). The effect of carbon sources was further analyzed based on

response to mineral and time. Additionally, we examined the main effect of the minerals

because there was a highly significant effect with an F value over 10 times greater than that

of the interaction. In this set of experiments, carbon-source amendment, in the absence of

mineral amendment, had only a slight effect on PFIL production at 2 and 5 days incubation

(Fig. 2).

o

00

o

COc

320)

>

su o ^

U> C U)

o « qh; o o

% _2r> .2

cô CD O O (3

Carbon-source amendment

Figure 2. Influence of carbon-source amendment (190 on production of PHL (A, B) and PLT (C,

D) by CHAO grown for 2 (A, C) and 5 (B, D) days, with (solid bars) and without (shaded bars)

zinc-sulphate (0.35 mM) and ammonium-molybdate (0.5 mM) amendments. Values represent the

mean of six cultures (+ SE).

Glycerol gave a slight increase and glucose almost completely repressed pyoluteorin

production at 2 and 5 days. However, PLT began to accumulate in glucose amended

cultures after prolonged incubation. This was not uniformly observed with the other carbon

sources suggesting that repression was transient and that PLT production resumed as the

glucose began to deplete.

When carbon sources were tested in the presence of mineral amendments, fructose,

mannitol, and glycerol increased PHL production at 2 and 5 clays and increased PLT

production at 5 days compared with the no carbon source control and with the carbon

source but no mineral controls. Incubation time had no consistent effect on PHL

52

production, but PLT production was greater after 5 days. Across all carbon sources, and

regardless of time, a mixture of zinc-sulphate and ammonium-molybdate significantly (P <

0.0083) increased production of PHL from 3.8 to 110.7 ng/10" CFU. and production of

PLT from 55.1 to 203.8 ng/10" CFU, compared with production in the absence of mineral

amendment.

Production of pyrrolnitrin was greater at 5 days compared with 2 days (P = 0.0267),

and there were significant interactions between carbon source x time (P = 0.0001) and

mineral amendment x time (P = 0.0187). Further analysis of these interactions indicated

that mineral amendment significantly increased pyrrolnitrin production at 2 days (P =

0.0278) from 11.6 to 17.1 ng/10" CFU and at 5 days (P = 0.0165) from 32.4 to 68.9 ng/10'

CFU, compared with production in the absence of mineral amendment. At 2 days, although

production was weak in all treatments, glycerol gave a slight but significant increase

compared with the control (Fig. 3). At 5 days, production was virtually unchanged in the

absence of carbon-source amendment

1 10Figure 3. Influence of carbon-source amendment

(\%) on production of pyrrolnitrin by CHAO

grown for 2 (A) and 5 (B) days. Values represent

the mean of six cultures (+ SE).

o <t>

o

2

W O O

Carbon-source amendment

or when sucrose, which is not utilized by CHAO. was supplied. When fructose or mannitol

were provided, pyrrolnitrin production was increased over 5-fold (Fig. 3).

Influence of minerals and carbon sources on siderophore production. Cobalt-

chloride was the only mineral which increased pyochelin, and ammonium-molybdate was

the only mineral which increased salicylic acid production in nonamended NBY broth

53

(Table 3). Copper and iron reduced pyochelin production and zinc increased salicylic acid

production in the presence of glycerol. None of the minerals had an effect on production of

either siderophore in media amended with glucose. Glucose used alone and in combination

with minerals generally increased pyochelin production, except in the presence of iron

when it reduced production. Glycerol used alone or in combination with minerals generally

did not give a significant increase in pyochelin. Wlien combined with dilute NBY broth,

however, glycerol gave a slight and glucose a dramatic increase in production of both

pyochelin and salicylic acid (Table 3). Combination of minerals with either glycerol or

glucose generally increased salicylic acid production compared with the minerals alone, but

had no influence on the effect of the carbon sources alone (Table 3).

We then evaluated the interactive effects of a larger range of carbon sources.

incubation time, and a mineral mixture of zinc-sulphate and ammonium-molybdate on

siderophore production. For pyochelin production, the highest order interactions that were

significant were carbon source x mineral (P = 0.0001) and carbon source x time (P =

0.0002). The effect of carbon source was further evaluated based on responses to mineral

and time. For salicylic acid production, the highest order interaction that was significant

was carbon source x mineral (P = 0.0064). The effect of carbon source was further

analyzed based on response to mineral.

Fructose, mannitol, and glucose significantly (P = 0.0001) increased pyochelin

production from approximately 56 ng/10s CFU for the nonamended control without

minerals to 302, 316, and 788 ng/10s CFU, respectively (Fig. 4). Sucrose and glycerol had

no effect on pyochelin production. Mineral amendment with zinc-sulphate and ammonium-

molybdate reduced pyochelin production four-fold in the presence of glucose but had no

effect on pyochelin production with other carbon sources. In contrast, mineral amendment

significantly (P = 0.0001) increased production of salicylic acid by two- to three-fold

regardless of carbon source amendment (Fig. 4). In the absence of minerals, carbon source

amendment had no effect on production of salicylic acid. With amendment of minerals,

mannitol and glycerol increased salicylic acid production and glucose reduced production

compared with the no carbon source control.

54

TABLE

3.Influenceofmineralsandcarbon-sourceamendmentonsiderophoreproduction

(ng/

10sCFU)by

P.fluorescensCHAO.

Pyochelin

Sali

cyli

cacid

NBY

NBY

NBY

NBY

NBY

NBY

Culturemedia

plus

glycerol

plus

glucose

LSD

plus

glyc

erol

plus

glucose

LSD

NBY

alone

56.7

104.9

185.4

68.2

<0.I

18.3c

18.6

11.2

CuSO,

24.6

25.1

1,002.2

567.9

11.4

49.3

17.2

86.3

ZnSO,

61.3

146.4

327.4

165.2

<0.1

75.5

37.4

53.7

CoCi

150.2

107.9

945.5

ns

<0.1

22.9

48.5

32.9

Mo,(

NH,),p,(

50.5

85.2

149.2

53.1

26.4

23.7

58.6

29.6

MnCl

74.7

72.3

170.7

87.1

<0.1

9.6

51.7

22.5

LiCl

56.6

89.0

204.9

91.4

<0.1

13.0

20.8

5.5

FeSO,

39.4

11.6

5.9

24.6

0.9

10.4

21.4

11.5

B(OH),

76.3

88.8

188

180.1

2.2

13.1

24.1

6.2

IVlg

SO,'

81.9

89.8

3620

172.6

8.2

12.6

46.8

26.5

NaCl

53.6

130.2

1972

92.0

1.2

20

7148

9.5

CaCl,

66.0

67.0

1395

43.7

8.4

8.8

22.4

11.8

DiluteNBY

<0.1

174.9

2,361

91,756.9

4.9

78.7

416.5

268.6

DiluteNBY

withNaCl

<0.I

273.4

4,6953

1,130.5

<0.1

68.6

2,848.2

992.9

LSD=

56.6

64.9

1,041.0

13.5

32.1

448.3

'

Within

arow,

foreachmedium,si

derophoreconcentrationwas

sign

ific

antl

ydifferentaccording

toFisher'sLSD

test(P

<0.0471

),exceptwhere

'ns'

indicatesanonsignificantANOVA.Withinacolumn,siderophoreconcentrationwas

sign

ific

antl

ydifferentaccording

totheLSD

atthebottom(P<

0.0172).

55

Figure 4. Influence of carbon-source amendment ( 1 %)

on siderophore production by CHAO. A, salicylic acid

production with (solid bars) and without (shaded bars)

amendment of zinc-sulphate (0.35 mM) and

ammonium-molybdate (0.5 mM); B, pyochelin

production with (solid bars) and without (shaded bars)

mineral amendments. Preliminary ANOVA found a

significant interaction between pyochelin production

and incubation duration. C, depicts further analysis of

pyochelin production after 2 (shaded bars) and 5 (solid

bars) days. Data with and without minerals were

pooled. Values represent means of six cultures (+ SE).

Influence of zinc, inorganic phosphate, and glucose on growth and

antibiotic production by diverse biocontrol strains. Strains varied in tolerance to

zinc-sulphate. All ARDRA 1 strains could be grown in media amended with 0.7 mM

zinc-sulphate without reduction in CFU after 48 h incubation; however, the maximum

concentrations for strains m ARDRA groups 2 and 3 before growth was significantly

reduced was approximately 0.2 mM. For determination of antibiotic production, 0.7 mM

zinc-sulphate was used for ARDRA 1 strains and 0.2 mM was used for all other strains.

Amendment with I % glucose increased the growth of all strains by 0.5 to 1 log1()

CFU/ml with no significant differences observed anions strains nor among ARDRA or

RAPD groups.

Most strains produced only a low amount of PHL in nonamended NB and

production was not correlated with either ARDRA or RAPD grouping in this medium.

Zinc-sulphate stimulated PHL production in CHAO and most other ARDRA 1 strains

(Table 4). Of all the ARDRA 2 and 3 strains only TMLA4 and Fl 13 were stimulated.

Zinc-sulphate slightly reduced PHL production in PITR2, but did not have a significant

impact on other strains (Table 4). There were slight but significant negative correlations

between increased PHL production by zinc-sulphate and ARDRA (P = 0.0005, r = -

0.31) and RAPD group (P = 0.0054. r = -0.25). Glucose increased PHL production by

all ARDRA 1 strains except PGNLi and by all ARDRA 2 strains except PITR2,

Carbon-source amendment

56

TABLE 4. Influence of zinc-sulphate and glucose on production of 2,4-diacetylphloroglucinol (ng/10sCFU) by biocontrol strains of Pseudomonas fluorescens in nutrient broth.'

Nutrient broth amendment

Strain None Zinc-sulphate Glucose LSD

CHAO 1.3 48.7 84.2 36.0

PF 0.7 271 9 224.8 151.6

Pfl 0.5 28.9 101.2 37.9

Pf5 1.9 315.5 290.3 246.7

PGNR1 1.0 46.3 78.6 52.0

PGNR2 0.6 15.7 22.4 14.3

PGNR3 LI 39.4 56.3 34.0

PGNR4 0.2 37.6 78.6 44.3

PGNL1 1.2 22.6 56.4 ns

PINR2 0.4 12.2 130.8 114.9

PTNR3 0.5 9.7 132.1 106.2

C*1A1 71.4 60.2 97.8 ns

CMLA2 31.0 83.1 143.0 ns

CALB2 22.0 8.8 101.3 69.0

PILH1 0.7 2.6 88.4 68.5

PITR2 82.6 6.4 82.8 62.2

PITR3 1.0 0.3 103.2 68.1

Ql-87 1.0 1.1 102.7 55.8

Q2-87 0.6 0.4 151.4 78.5

Q4-87 1.6 0.7 97.5 23.6

Q5-87 0.8 1.2 162.7 69.9

Q6-87 0.5 5.1 150.8 52.4

Q7-87 0.7 1.0 129.8 87.7

Q8-87 0.1 22.1 174.0 74.8

Q9-87 0.1 2.1 168.4 79.1

Q12-87 0.9 1.0 162.1 77.7

Q13-87 0.5 0.6 129.8 122.9

Q37-87 0.9 1.7 187.9 142.8

Q65-87 12.9 58.4 122.2 62.3

Q86-87 0.4 1.8 169.7 118.2

Q88-87 0.1 0.1 134.0 85.6

Q95-87 10.4 3.9 95.2 19.0

Q112-87 4.2 8.1 102.9 53.4

Q128-87 46.9 46.8 167.6 ns

Q139-87 3.1 2.9 199.6 99.5

TM1A3 5.1 1.1 86.4 57.4

TMLA4 17.6 108.3 112.0 70 0

TM1A5 4.7 1.7 J 37.3 78.4

TMLA5 55.6 97.0 123.0 ns

TM1B2 51.4 96.0 120.1 ns

P12 1.4 1.5 52.5 42.2

Fl 13 9.9 58.6 12.0 13.5

LSD= 19.4 52.3 108.3

57

ARDRA 1 0.8 77.2 114.2

ARDRA 2 14.8 21.5 131.2

ARDRA 3 5.6 30.0 32.3

42.3

13.5

ns

LSD= 16.2 49.1 53.8

'

For each amendment, the main effects of strain and ARDRA group were significant at P = 0.050 and P

= 0.008, respectively, except where 'ns' indicates a nonsignificant ANOVA test for amendment with no

LSD test applied.

C*l Al, CM 1 'A2, and Q128-87 (Table 4). There was no correlation between glucose

and ARDRA group. Strains in ARDRA groups 1 and 2 had similar positive responses to

glucose. In contrast, only one of the two ARDRA 3 strains. P12 from tobacco in

Switzerland, was stimulated by glucose.

Only the ARDRA I strains produced PLT and data from the other strains were

not included in the analysis. Among the ARDRA 1 strains, the quantity of PLT

produced varied in NB and NB amended with zinc-sulphate (Table 5). Strains PF and

Pf-5, the only ARDRA 1 strains in RAPD group 2, were the most productive in both

TABLE 5. Pyoluteorin production by Pseudomonas fluorescens biocontrol strains in

nutrient broth with and without zinc sulphate amendment.'

Strain

Pyoluteorin (ne/1(2 CFU)

None Zinc-sulphate LSD

13.3 66.5 36,3

39.8 274.7 128.0

29.9 144.7 102.8

39.7 408.7 294.2

13.7 32.6 17.4

11.3 31.7 13.7

15.3 63.0 40.3

12.7 48.8 7.2

16.0 41.1 20.9

15.9 14.2 ns

25.1 69.3 ns

CHAO

PF

Pfl

Pf5

PGNR1

PGNR2

PGNR3

PGNR4

PGNL1

PINR2

PINR3

LSD=: 3.0 110.6

'

Within a row, for each strain, pyoluteorin concentration was significantly greater in nutrient

broth amended with 0.7 mM zinc-sulphate (P = 0.0391) except where 'ns' indicates a

nonsignificant ANOVA with no LSD applied. Within a column, strains varied significantly in

pyoluteorin production (P = 0.0003).

58

media. Zinc-sulphate amendment significantly increased PLT production by most

strains by 3- to 7-fold compared with production in nonamended media. The only

strains which did not have a significant response to zinc-sulphate were PINR2 and

PTNR3, the only ARDRA 1 strains isolated from Albenga soil from Italy (Table 5). In

contrast, glucose reduced pyoluteorin production by all strains to below the detection

limit.

Inorganic phosphate inhibited PFIL production by strains in all ARDRA groups,

but to varying degrees (Table 6). For example. PHL production by CFIAO was almost

abolished by 10 mM phosphate, while 100 mM phosphate reduced production by Q2-87

by only 10 fold (Table 6). No strain was insensitive to 100 mM phosphate. Production

of PLT by CHAO was completely inhibited by 100 mM but only slightly reduced by 10

mM phosphate (data not shown). Pyrrolnitrin production by CHAO was not affected by

200 mM phosphate (data not shown). Bacterial growth was generally increased 5 to 10

fold by 100 mM phosphate amendment (data not shown).

TABLE 6. Phosphate-repression of 2,4-diacetylphloroglucinol production (ng/108 CFU) byPseudomonas fluorescens biocontrol strains.'

Phosphate amendment

Strain None 10 mM lOOmM

CHAO 35.4 ± 8.9 1.1 ± 0.5 0

Pf5 252.7 ± 49.9 nd 28.1 ± 9.9

PITR3 279.01 38.1 nd 31.8 221.3

Q2-87 108.4 2 8.6 nd 13.2 ± 3.4

Q65-87 164.3+ 29.1 nd 2.1 ± 0.4

TM1A3 276.4+ 74.7 nd 1.9+ 0.4

TM1A5 271.9 2102.7 nd 1.9 ± 0.7

P12 100.5 ± 21.6 nd 0

F113 550.8 2 51.4 nd 5.1 ± 2.3

'

Bacteria were grown 48 h m NB plus 17c glucose, except Fl 13 grown m NB plus 1% sucrose.

Antibiotic yield was determined with HPLC as described in Materials and Methods, and

expressed as ng/10" CFU. Values represent the means of three trials ± standard error. nd=not

determined.

59

Discussion

Bacterial gene expression in the rhizosphere is regulated both by endogenous and

exogenous signals. Exogenous regulatory signal(s) activate LemA, a membrane-bound

sensor-kinase, which in turn regulates production of bacterial autoinducers that control

biosynthesis of antibiotics critical for the biocontrol of soilborne fungal pathogens

(Pierson et al. 1988). However, such signals have not yet been determined. Using a

liquid culture assay, we identified several putative environmental signals that influenced

production of antifungal metabolites by an ecologically diverse collection of biocontrol

strains.

We observed that carbon sources commonly found in plant root exudates had a

differential influence on the spectrum of antibiotics produced by individual biocontrol

strains irrespective of their effects on bacterial growth. For example, production of PLT

and PHL by strain CHAO was stimulated by glycerol and glucose, respectively.

Environmental conditions influencing PHL production generally had the same effect on

monoacetyl-phloroglucinol which provides further evidence that this is a precusor

compound (Shanahan et al. 1993). Glucose, however, repressed PLT, with antibiotic

accumulating only after prolonged growth when glucose began to deplete. Evidence

suggests that glucose may block antibiotic production through repression of

dehydrogenases that catalyze glucose oxidation, a reaction that transfers electrons from

the enyzyme co-factor PQQ to the electron transport chain (Gutterson 1990). A PqqF-

mutant of CHAO, which lacks glucose dehydrogenase activity, over-produces

pyoluteorin (Schnider et al. 1995). We confirmed that CHAO also produces pyrrolnitrin

in the presence of fructose and mannitol, albeit after incubation times considerably

longer than are typically used to monitor PHL and PLT production, and at

concentrations much lower than other biocontrol strains (e.g., Pf-5) (B. Duffy

unpublished data. Sarniguet et al. 1995). W2eak production by this strain reflects

competition for the common substrate L-tryptophan m the pyrrolnitrin and indole-3-

acetic acid biosynthetic pathways (Kirne r et al. 1998, Oberhänsli et al. 1991). Although

carbon sources differentially influence media acidification during bacterial growth

(Dekleva and Strohl 1987), which may have an indirect effect on antibiotic production

60

(Slininger and Shea-Wilbur 1995) and biocontrol activity (Ownley et al. J 992), we did

not observe such pH changes with media amendments used in this study.

Plant specificity of biocontrol strains has been demonstrated at both the species

and cultivai- level (Maurhofer et al. 1995. Smith et al. 1997. Weller 1988). This has

generally been attributed to differential utilization of the various carbon and nitrogen

compounds found in exudates and its effects on bacterial growth and population

structure (Lemaneeau et al. 1995, O'Connell et al. 1996, Westover et al. 1997). Our

results suggest that another factor in plant specificity may be the influence of root

exudate components on the biosynthesis of antimicrobial metabolites. Quantitative

and/or qualitative differences in the composition of root exudates could determine the

predominant biocontrol mechanism expressed in given crop-pathogen systems. This

would clarify results of genetic studies that have demonstrated a role for PHL (but not

PLT) in biocontrol on wheat and cucumber, and a role for PLT on cress and cotton

(Loper et al. 1997, Maurhofer et al. 1994). Using an ice-nucleation reporter gene

system, Kraus and Loper (1995) recently observed differential expression of PLT

biosynthetic genes in P. fluorescens Pf-5 on cotton and cucumber seed.

Differences were also observed in the production of particular antibiotics by

diverse strains. For example, glucose stimulated PHL production in almost all of the 42

strains screened, with a notable exception of P. fluorescens FT 13 from Ireland. In this

strain, sucrose stimulated PHL production but glucose had no effect, confirming

previous observations of Shanahan et al. (1992). Incidentally, Fl 13 was the only strain

isolated from sugarbeet, the roots of which tend to have an unusually high sucrose

content. This suggests that evolutionary relationships may exist between biocontrol

strains and their original host plants, and further supports the notion of breeding for

closer plant-biocontrol agent interactions to achieve improved disease suppression. A

natural example of such selection is decline of take-all disease due to the accretion of

PHL-producing Pseudomonas spp. in the soil and rhizosphere after wheat monoculture

(Raaijmakers et al. 1997). Many of the strains used in this study were isolated from a

take-all decline soil in the USA (Keel et al. 1996).

We further demonstrated a differential effect of minerals on antibiotic

biosynthesis. Zinc-sulphate stimulated production of both PHL and PLT by diverse

strains, while ammonium-molybdate stimulated only PHL, and cobalt-chloride

61

stimulated only PLT. Pyrrolnitrin production was stimulated by a mixture of zinc and

ammonium-molybdate. Other sulphate and chloride compounds did not have this effect

indicating that Zn, Co, and NH,-Mo were the active cations. Inorganic phosphate

repressed PFIL and to a lesser extent PLT but had no effect on pyrrolnitrin. Phosphate

repression has been reported for other polyketide antibiotics (e.g., anthracycline and

tetracycline) and phenazines in Pseudomonas (Martin et al. 1994, Turner and Messenger

1986), and zwittermycin A and kanosamine in Bacillus (Millier et al. 1995, 1996). and

may be a common phenomenon in soil bacteria. Strains, however, differed in sensitivity

to phosphate repression. This explains why Keel et al. ( 1996) detected PHL production

in some but not all strains on King's medium B which contains approximately 9 mM

K,HP04. Interestingly, iron, which stimulates production of a variety of antifungal

metabolites Leg., zwittermycin A (Milner et al. 1995), kanosamine (Milner et al. 1996),

phenazine (Slininger and Jackson 1992). and cyanide (Keel et al. 1989)], affected

neither PFIL nor PLT. This does not exclude a role for iron in biosynthesis since trace-

amounts found in NBY and on glassware may have been sufficient. How minerals

influence antibiotic production by biocontrol pseudomonads is uncertain. In other

bacteria, minerals repress antiobiotic synthases, interupt transcription and promotion of

biosynthetic genes, and may indirectly affect nutrient availability and pH (Behal and

Hunter 1995, Cousins 1994, Martin et al. 1994). Also, zinc and other minerals are

essential for growth, they influence cell membrane integrity, and are key

components/catalysts of over 300 enzymes and other proteins (Weinberg 1977). It has

been suggested that increased antibiotic biosynthesis is a response to environmental

stress conditions (e.g., phosphate starvation, heavy-metal toxicity) that decrease

bacterial growth (Behal and Hunter 1995, Martin and Demain 1980). Further study to

confirm this is particularly relevant to biocontrol bacteria introduced into soil where

conditions can be extreme.

From a practical perspective, mineral effects on antibiotic biosynthesis may

explain the association between soil chemical and physical properties and the variable

performance of biocontrol strains between field sites (Duffy unpublished data.

Thomashow and Weiler 1996). For example, zinc which stimulated antibiotic

production in CHAO is typically more abundant in the naturally disease suppressive

soils from which this strain was isolated, and CHAO is not effective when added to

62

disease conducive soils that contain less zinc (Défago and Haas 1990). Ownley and

coworkers (Thomashow and Weiler 1996) similarly found that zinc soil content (DTPA-

cxtractable) was positively correlated with the biocontrol activity of P. fluorescens 2-79.

Independently, Slininger and Jackson (1992) demonstrated that zinc stimulated

production of phenazine-1-carboxylate. the primary biocontrol determinant in strain 2-

79 (Thomashow and Weiler 1996). In contrast, we found no effect of zinc on PHL

production by Q2-87, a strain for which biocontrol was not correlated with zinc soil

content (B. Duffy, B. Ownley, and D. Weiler, unpublished data). Identifying factors

favorable to biocontrol will facilitate the targetted deployment of specific strains and

strain mixtures in field locations more suitable to their activity, so-called 'prescription

biocontrol' (Cook 1993). Duffy et al. (1997) identified soil factors favorable to take-all

suppression by Trichoderma koningii and applied this information to develop a model

enabling its performance to be predicted at field sites in the USA and China. Another

potential application of our work is the development of mineral amendments to improve

biocontrol under unfavorable conditions. Preliminary work has shown that zinc-EDTA

amendments improved biocontrol of Gaeumannomyces graminis var. graminis by 2-79

in a zinc-deficient soil (Thomashow and Weiler 1996). Our finding that biocontrol

strains differ in zinc tolerance, however, emphasizes the need to minimize potential

toxicity to other beneficial microorganisms. Providing minerals directly in biocontrol

formulations would reduce total dosage applied to the environment and optimize

availability to the target agent. Our finding that inorganic phosphates repress antibiotic

production by diverse strains raises important questions about potential adverse effects

of phosphate fertilizers commonly used in agriculture on not only introduced biocontrol

agents but also indigenous populations of antagonists.

Modulating the production of antimicrobial metabolites during growth may also

improve the quality of inoculants. Lowering PHL and PLT concentrations in inoculants

with phosphate amendments would avoid potential phytotoxicity problems (Maurhofer

1994, Slininger et al. 1996, Thomashow and Weller 1996), and at the same time

increase bacterial growth (Martin et al. 1994). On the other hand, increasing antibiotic

concentrations may provide a bridge of protection against diseases with a rapid-onset

(e.g., Pythium damping-off) that outpace the ability of introduced bacteria to establish in

the rhizosphere and commence in situ antibiotic production. Zinc and other minerals

63

have the extra benefit of improving genetic stability in inoculants (Duffy and Défago

1995). We have identified a number of mineral and/or carbon-source amendments that

stimulate siderophore production in P. fluorescens. Siderophores, particularly salicylic

acid, have been implicated in the ability of certain strains to trigger induced resistance in

plants (De Meyer and Höfte 1997, Maurhofer et al. 1998), and increasing their supply

via inoculants may be advantageous. Zinc has previously been reported to stimulate

production of pyochelin and pyoverdin in P. aeruginosa biocontrol strain 7NSK2 (Höfte

et al. 1994) and plant-associated Az.otobacter vinelandii (Huyer and Page 1989).

Interestingly, zinc stimulation relieves bacterial siderophore production from iron-

repression (Höfte et al 1994), which might allow a greater role for siderophores in

microbial interactions under iron-sufficient conditions (Loper and Henkels 1997).

Identifying differential responses to signals sheds new light on the regulation of

antibiotic biosynthesis and its evolution. By screening strains together we avoided

differences that could be attributed to variations in experimental conditions in different

laboratories working with single strains. The strains we studied have a conserved

biosynthetic region (ph/D) for antibiotic production (Keel et al. 1996), but are

genetically different and have been characterized into three ARDRA and seven PCR-

RAPDs groups (Keel et al. 1996). Responses to zinc and glucose were not linked to any

particular group of strains which may reflect adaptation to specific local conditions. We

recently reported that fusaric acid repression of PHL is ARDRA group dependent

(Duffy and Défago 1997b) suggesting this is a more general adaptation. At this point we

cannot say whether adaptations occurred in signal uptake/recognition, global activation,

autoinduction, biosynthetic gene promotion, or antibiotic processing. Export was not a

factor though since our extraction procedures involved cell lysis and the release of

intracellular antibiotics. Sequencing the phi Operon and flanking regions of several

strains will shed more light on environmental regulation: currently the complete

sequence is available only for Q2-87 (Bangera and Thomashow 1996). Relieving strains

or making them more responsive to certain environmental signals has been exploited for

increased antibiotic production in pharmaceutical fermentations (Martin and Demain

1980), and we believe it presents new opportunities to improve biocontrol. Our results

with PHL suggest that screening strain collections for differential responses to

environmental signals, may also be a useful approach to improve the biocontrol activity

64

of bacteria which carry the highly conserved biosynthetic loci for phcnazine and

zwittermycin A (Stabb et al. 1994, Thomashow and Mavrodi 1997).

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70

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71

Chapter 3

Zinc Improves Biocontrol of Fusarium Crown and Root Rot of Tomato by

Pseudomonas fluorescens and Represses the Production of Pathogen

Metabolites Inhibitory to Bacterial Antibiotic Biosynthesis

Abstract

Tomato crown and root rot caused by Fusarium oxYsporum f. sp. radicis-lycopersici (FORL) is

an increasing problem in commercial production in Europe. Tsrael, Japan, and North America.

Widely effective Pseudomonas fluorescens biocontrol strain CHAO provides only moderate

control of this disease. A one-time amendment of 33 ug/ml zinc (added as EDTA salt complex)

to hydroponic nutrient solution did not reduce disease when used alone but did improve

biological control with CHAO by 25% in soilless rockwool culture. Zinc at concentrations as

low as 10 pg/ml abolished production of the phytotoxin fusaric acid, a Fusarium pathogenicity

factor, and increased conidia production over 100 fold, but reduced total fungal biomass.

Copper (33 ug/ml Cu:+ added as EDTA salt) had a similar effect on the pathogen, reduced

disease when used alone, and had an additive effect on biocontrol with CHAO in soilless culture.

Ammonium-molybdate did not improve biocontrol nor affect production of fusaric acid or

conidia. Strain CHAO did not degrade fusaric acid. Rather, fusaric acid at concentrations as low

as 0.12 pg/ml repressed production by CHAO of the antibiotic 2,4-diacetylphloroglucinol, a key

factor in the biocontrol activity of this strain. Production of pyoluteorin was also reduced, but

hydrogen cyanide and protease were not affected suggesting an activity for fusaric acid at the

biosynthetic level or at a regulatory level downstream of gacA and apdA, Fusaric acid did not

alter nor interfere with the recovery of preformed antibiotics and bacterial growth was not

affected by fusaric acid at concentrations as high as 200 pg/ml. We suggest that zinc, which did

not alleviate repression of antibiotic biosynthesis by fusaric acid, improved biocontrol by

reducing fusaric acid production by the pathogen thereby increasing antibiotic production by the

biocontrol agent. Indeed, when microbial metabolite production was measured in the rockwool

bioassay. zinc amendments reduced fusaric acid production and enhanced 2,4-

diacetylphloroglucinol production. This demonstrates that pathogens can have a direct negative

impact on the efficacy of biocontrol agents at a mechanism level.

Published as Phytopathology 87:1250-1257.

72

Micro- and macroelement amendments have been commercially used on a limited scale

to manage certain soilborne diseases, including Fusarium wilt of tomato and other

vegetable crops (Engelhard 1989). Disease reduction is most often attributed to

improved nutrition that boosts host defenses and/or to direct inhibition of fungal growth

and activity. Pathogen suppression may also be an indirect result of amendment-

mediated modification of chemical and physical properties like soil and rhizosphere pH

(Simon and Sivasithamparam 1989) or modification of host root exudates to disfavor

pathogenic activity (Htiber 1989). In a few cases though, mineral amendments appear to

reduce disease by indirectly stimulating indigenous populations of microorganisms that

are beneficial to plant growth and antagonistic to pathogens (Htiber 1989). For example,

broadcast application of NaCl, which has traditionally been used to manage E^usarium

on asparagus, favors populations of manganese-reducing bacteria in the rhizosphere that

increase availability of this essential trace-mineral to the plant and may evince a

fungistatic effect on the pathogen (Elmer 1995). While exploitation of indigenous

microbial communities is an ecologically sound approach to achieve sustainable disease

control and deserves greater attention, this approach relies on germinal populations that

may not be present in all soils. It also may require several growing seasons to obtain

economic control, and may not be compatible with all cropping systems.

There is increasing interest in the introduction of bacterial and fungal biocontrol

agents for managing soilborne diseases, partly as a response to public concerns about

nontarget effects of synthetic pesticides and fumigants but also because of a lack of

effective compounds for soilborne diseases (Cook 1993). However, many biocontrol

agents are inconsistent in their performance from site to site, and this has been a primary

obstacle to commercial development (Weiler and Thomashow 1994). Soil chemical and

physical properties have been implicated in the variable biocontrol activity of

Pseudomonas fluorescens strain 2-79 and Frichoderma koningii against take-all on

wheat (Duffy et al. 1996. Weller and Thomashow 1994), and results indicate that

introduced biocontrol agents and indigenous populations of antagonistic microbes are

influenced by some of the same abiotic soil conditions. WThile effects of pH, and to a

lesser extent clay type, organic matter content, and organic amendments on biocontrol

agents have been reported (Ownley et al. 1992. Slininger and Shea-Wilbur 1995. Taylor

and Harman 1990. Voisard et al. 1994. Voisard et al. 1989), the influence of minerals on

73

suppression of soilborne disease has received little attention and the potential of mineral

amendments for optimizing biocontrol remains largely unexplored.

In this study we evaluated the utility of ammonium-molybdate, copper, and zinc

trace-mineral amendments as an approach to Improve the biocontrol activity of

Pseudomonas fluorescens strain CHAO. This strain was isolated from roots of tobacco

grown in soil, collected near Morens in western Switzerland, that is naturally

suppressive of black root rot caused by Chalara elegans (synanamorph Thielaviopsis

basicola). Application of CHAO to seed or plant growth medium effectively controls

this and a variety of other soilborne fungal diseases, but the level of control can depend

on the predominant type of clay mineral and on the pathosystem (Voisard et al. 1994).

The primary mechanism of biocontrol for most diseases is the production of the

antimicrobial compounds 2.4-diacetylphloroglucinol (PHL). pyoluteorin, and hydrogen

cyanide (Voisard et al. 1994). Strain CHAO produces several high-affinity metal-

chelating siderophores (i.e., pyoverdinc pyochelin. and salicylic acid) that may

contribute to nutrient competition with pathogens and to systemic acquired host

resistance (Voisard et al. 1994). Recently, we have found that in vitro production of

PHL and pyoluteorin is stimulated by zinc, and production of salicylic acid is stimulated

by molybdenum and magnesium (Duffy and Défago 1996) while iron availability is

critical for hydrogen cyanide production (Voisard et al. 1994).

To test our hypothesis that mineral amendments will improve the biocontrol

activity of CHAO. we selected tomato crown and root rot caused by Fusarium

o.xysporum Schlechtend.:Fr. f.sp. radicis-lycopersici Jarvis & Shoemaker (FORL) as our

model pathosystem. This is an increasingly important disease in commercial tomato

production in Europe, North America, Japan, and Israel and losses are especially severe

at the seedling stage in soilbed and hydroponic culture (Mihuta-Grimm et al. 1990).

Minerals were evaluated in a rockwool soilless bioassay because this was more

amenable than soil to management of nutrient supply, but also because this is the

predominant method for tomato production in Europe. Our objectives were (i) to

determine the level of control provided by CHAO against tomato crown and root rot. (ii)

to determine the influence of zinc, copper, and ammonium-molybdate amendments on

disease and the biocontrol activity of CHAO, and (iii) to elucidate possible mechanisms

74

of action for minerals by observing their influence on the pathogen and the biocontrol

agent.

Materials and Methods

Microorganisms and inoculum production. FORL strain 22 (gift of C. Alabouvette,

INRA, Dijon, France) was rcisolated from infected tomato and stored on 2% malt agar

at 3 °C and in 2% malt broth (pH 5.5) plus 40% glycerol at -80 °C. Starter cultures of

malt broth (150 ml per 500 ml Erlenmeyer flask with one baffle. A. Dumas, Zürich)

were inoculated with six 4-mm agar plugs taken from a fresh malt agar culture.

Inoculum was produced by inoculating malt broth with 1 ml of a starter culture.

Cultures were incubated 10 to 14 days at 24 °C with 150 rpm. Fungal biomass (mycelia

and microconidia) was collected by ccntrifugation at 4,000 rpm for 15 min then briefly

homogenized in a blender.

Wild-type CHAO, spontaneous rifampicin resistant derivative CHA0KU, and

antibiotic overproducing derivative CHA0/pME3424 were stored in Luria-Bertani broth

plus 40% glycerol at -80 °C. Wild-type CHAO and the rifampicin resistant derivative

were similar for growth in Luria-Bertani broth, carbon source utilization (Biolog.

Hayward, CA), production of PHL and PLT antibiotics, and FORL growth inhibition in

vitro, Inoculum was produced in 150 ml LB in 500 ml Erlenmeyer flasks incubated 24 h

at 27 °C with 140 rpm. Bacteria were collected with centrifugation. washed once with

sterile bidistilled water, and brought to a concentration of approximately 109 CFU/ml.

Influence of trace-mineral amendments on disease suppression in rockwool

soilless culture. Tomato seeds (Lycopersicum esculentum Mill. 'Bonnie BesF; gift of

Peptoseed Co., Saticoy, CA) were surface disinfested for 30 min in 1% sodium

hypochlorite and pregerminated for 48 to 72 h on 0.852c water agar at 24 °C in

darkness. Seeds with radicles 2 to 4 mm long were placed in the dipples of rockwool

cubes (3.5 cm2 diameter x 4 cm deep: one seed per cube: Grodania A/S, Hedehuscne,

Denmark) with 18 cubes per plastic tray (23.5 x 28.5 cm diameter x 5.5 cm deep). The

rockwool was saturated with I liter of a nutrient solution designed for commercial

hydroponic tomato production by the Office of Horticultural Production of Geneva.

Switzerland and was composed of (mg/liter): Ca(NO,)~x 4H,0, (955.5): NFI,H,P04,

75

(660); KNOv (450); MgS04x 7H20, (360); K2S04, (174); KH2P04, (78); Fe-EDDHA,

(25); EDDHA, (4); Na2B407x 10PL/O, (1.05); MnSO(x 7H20, (1.02); ZnS04x 7H;0,

(0.43); CuS04x 5H20, (0.2); Na2Mo04x 2H:0, (0.04). Prior to saturating the rockwool,

the nutrient solution was inoculated with CHAO at 2 x 10' CFU/ml and/or inoculated

with FORL at 4 x 10" microconidia plus mycelial fragments per ml. Filter-sterilized

stock solutions of minerals were added to give concentrations of 33 jig/ml zinc added as

EDTA disodium salt (Zn C10HI2RNa2Os x 4H20). Copper added as EDTA disodium salt

(Cu C10H]2N2Na2Os), or Mo7(NH,)(024x 4PLO. A mineral mix consisted of 1/3 the

concentration of each compound. Plants were grown in a growth chamber with J 6 h

light: 8 h darkness. 22 °C "day': 18 °C -night', and 70% RH. After 7 days,

approximately 400 ml of nutrient solution (without additional mineral, bacterial,

pathogen treatment) was added to each tray; otherwise millipore-filtcred bidistilled

water was added as needed to maintain a solution level of 1 to 2 cm. Fourteen to sixteen

days after planting, tomato seedlings were carefully removed from the rockwool with

the upper 1.5 cm of the root system attached. Fresh plant weight was measured and

disease severity was rated on a scale of 0 to 4 [adapted from Mihuta-Grimm et al.

(1990)] where 0=symptomless; l=slight brown discoloration of the upper root system;

2=moclerate brown discoloration of two-thirds or less of the upper root system;

3=extreme brown discoloration of the upper root system and numerous necrotic lesions

extending up the crown and stem; 4=seedling dead or nearly so. A representative sample

of brown tissue was plated onto Komada's medium (Komada 1975) to confirm FORL as

the cause of symptoms. Biocontrol activity was expressed using a suppression index

calculated as the disease rating for the FORL alone treatment minus the FORL plus

CHAO treatment. Treatments consisted of 18 plants and were replicated four times.

Treatments were arranged as a 5 x 3 factorial in a split-plot design with a main plot of

mineral treatment (none, zinc, copper, ammonium-molybdate, mineral mix) and a

subplot of biocontrol treatment (no microbial inoculants, FORL alone, FORL plus

CHAO).

Influence of minerals on pathogen growth, conidia production, and fusaric

acid production. Czapek-Dox medium (Oxoid. Flampshirc, U.K.; 20 ml in 100 ml

flasks with one baffle) amended with a ranae of mineral concentrations was inoculated

76

with 10 pd of a FORL starting culture to give approximately 10 to 100 microconidia per

ml and incubated for 8 days at 24 °C with 120 rpm. Numbers of microconidia were

determined using a haemocytometer and then cultures were centrifuged to collect total

fungal biomasss (mycelia plus microconidia) for dry weight determination after

lyophilization. The supernatant was acidified to pH 2 with 2 N HCl, mixed with 20 ml

ethylacetate, and shaken for 30 min at 200 rpm. The solvent phase was separated with

centrifugation at 4,500 rpm for 10 min and flash evaporated. The residue was

resuspended in 1 ml methanol and analyzed with HPLC using a Hewlett Packard 1090

liquid Chromatograph equipped with a reverse-phase column (4 x 100 mm) packed with

Nucleosil 120-5-C18 and thermostatically controlled at 50 °C. Samples of 10 til were

eluted at a flow rate of 1 ml/min with a three-step linear gradient of methanol in o-

phosphoric acid. Fusaric acid was detected with an ultra-violet diode-array detector at

270 nm at a retention time of approximately 5.1 min and quantified against a standard

curve of synthetic FA (mol. wt. 179.2; Sigma. St. Louis, MO). The recovery efficiency

of this extraction method was approximately 60% for synthetic FA added to media and

incubated for 2 days. Treatments were arranged in separate randomized complete blocks

for each mineral (zinc, copper, ammonium-molybdate, mineral mix) at four

concentrations of 0, 10, 33 and 100 jig/ml. Each treatment consisted of four replicate

flasks per trial.

Influence of fusaric acid on bacterial growth and secondary metabolism.

The relationship between FA concentration and repression of PHL and

monoacetylphloroglucinol was observed by inoculating 10 jitl of an overnight LB

culture of CHAO (K)" CFU/ml) into 100 ml wide-mouth Erlenmeyer flasks (sealed with

3.5 cm diameter cotton plugs) containing 20 ml PCG liquid medium (10 g bacto-

peptone, 1 g Casamino Acids, 10 g glucose, 1 liter bidistilled water, pH 6.5 after

autoclaving) (Toyoda et al. 1988) amended with FA at 0, 0.01, 0.04, 0.12, 0.37. 1.1, 3.3,

10, 100. and 200 (ig/ml. A fresh stock solution of synthetic FA was prepared

immediately prior to use by dissolving 200 mg FA in 1.5 ml methanol, bringing to a

volume of 8 ml with sterile bidistilled water, adjusting the pH to 6.5 with a few drops of

2 N NaOH. and sterilizing with a 2 |dm filter. Production of PHL was observed in PCG

medium. After 48 h incubation at 27 °C with 140 rpm. culture pH was measured and

77

bacterial growth was determined by spreading appropriate dilutions onto King's

medium B agar. Cultures were then acidified to pH 2 with 2 N HCl and extracted with

20 ml ethylacetate. Concentrations of PHL (mol. vvt. 210, retention time 11.6 min) and

monoacetylphloroglucinol (mol. wt. 186, retention time 6.2 min) were determined with

HPLC analysis with maximum detection at 270 and 290 nm, respectively (Keel et al.

1992). Effects of zinc (65 tig/ml zinc as ZnS04x 7 HO) on PHL production by CHAO.

of zinc on antibiotic repression by FA, and of FA (100 pg/ml) on PHL production by

the antibiotic over-producing derivative CHA0/pME3424 (Schnider et al. 1995) were

tested in PCG medium. Production of hydrogen cyanide was estimated with indicator

paper (Voisard et al. 1989) after 24 h growth in GNB media (8 g Difco nutrient broth, 4

g Difco bacto-gelatine, 4 g NaCl, 1 liter bidistilled water. pH 6.7 after autoclaving)

amended with 100 (ig/ml FA. Production of pyoluteorin (mol. wt. 268; retention time

9.4 min), pyochelin (mol. wt. 325; retention times for characteristic double peaks 10.1

and 10.8 min), and salicylic acid (mol. wt. 138; retention time 8.2 min) after 2 days

growth in GNB media was quantified with HPLC analysis at 313, 254, and 300 nm,

respectively, against standard curves of reference compounds. Production of

extracellular protease was determined after 5 days growth in GNB media with the

azocasein reaction (Sacherer et al. 1995). Treatments were arranged in a randomized

complete block design with three replicate broths per trial. Degradation of FA by CFIAO

was determined with HPLC analysis of extractions of 48 h PCG and GNB cultures with

and without 100 |ag/ml FA.

Influence of fusaric acid on the recovery of preformed antibiotic. To

determine whether the reduction in PHL accumulation in treatments amended with FA

may actually be due to complexing of the antibiotic or other alterations interfering with

antibiotic recovery rather than inhibition of biosynthesis, a 300 ml culture of CHAO

grown 48 h in PCG media was partitioned into three aliquots of 100 ml. One aliquot

was extracted and analyzed with HPLC to determine the concentration of PHL in the

culture. The other two aliquots were amended with 100 and 500 pg/ml synthetic FA.

incubated with shaking for 4 h at 27 °C, then extracted and analyzed with HPLC to

determine the concentration of PHL. Each treatment consisted of three replicate

cultures.

78

Metabolite production in the rockwool bioassay. Tomato plants were grown

in rockwool and FORL, CHAO, and Zn-EDTA were added to nutrient solution as

described above. After 2 to 3 weeks, tomato shoots were removed and the rockwool

with roots from two trays of 42 plants each was mashed by hand in a wide-mouth 5 liter

glass flask. Appropriate dilutions of the suspension were spread onto Komada's agar

and onto King's B agar plus 100 ug/ml actidione and rifampicin in order to estimate

numbers of FORL and CHAO'", respectively. The rockwool and nutrient solution

mixture was brought to pH 2.5 with 2 N HCl and then extracted with 2.5 liters of ethyl-

acetate for two periods of 5 to 10 min with a 1 to 2 hour stationary period between.

After extraction, the rockwool was discarded and the organic phase was transferred to a

graduated cylinder and kept overnight at 2 °C in darkness. Four aliquots of 250 ml each

were flash evaporated and the combined residues were concentrated in 1 ml methanol.

Extracts were kept at -20 °C for 2 to 5 days then centrifuged to remove precipitates.

Samples were analyzed with HPLC using a 4 x 250 mm reverse-phase column. The

recovery efficiencies for this procedure were approximately 30% for PHL and 45% for

FA estimated by adding reference compounds to rockwool with tomato plants and

nutrient solution. Retention times for reference compounds were approximately 22.5

min for PHL and 9.5 min for FA. Detection limits were approximately 0.07 ug/plant for

PHL and 0.45 ug/plant for FA with a 10-ul-injection volume. Each extraction consisted

of two trays of tomato (84 plants total) per treatment with three extractions made to

estimate metabolite production.

Statistical analysis. All experiments were repeated and data from two or three

trials were pooled for final analysis after confirming homogeneity of variances with an

F test and/or after finding no significant treatment x trial interaction in a preliminary

ANOVA. Main effects and interactions were analyzed for significance using the SAS

general linear models procedure (release 6.08; SAS Institute, Cary, NC) and means were

compared using Fisher's protected (P < 0.05) LSD test when appropriate. Relationships

between minerals with pathogen growth, and between FA concentration and antibiotic

production by CHAO were analyzed using the SAS regression procedure (Pearson

option) after log transformation to normalize the data. Microbial CFU data were

normalized with the log,,, + 1 transformation prior to analysis. For the in situ metabolite

79

production experiment, metabolite concentration data are presented with ranges from

three extractions.

Results

Effect of mineral amendments on tomato crown and root rot and on disease

suppression by CHAO. Introduction of FORL into rockwool seeded with tomato

resulted in severe crown and root rot (disease rating 3 on a scale of 0 to 4) (Fig.l) and a

50% reduction of plant fresh weight (Fig. 2) (P=0.000l). The pathogen was recovered

from all lesions plated onto Komada's medium. The main effects of mineral and

biocontrol treatment and the interaction had a significant influence on disease severity

rating (P=0.0001) and on fresh plant weight (P<0.0132). Data were further analyzed

based on the response to minerals and to biocontrol treatment.

In the presence of FORL, CHAO alone provided a moderate level of protection,

reducing disease from a severity rating of 3 to 2.5 (Fig. I); however, plant fresh weight

was not significantly increased (Fig. 2) (P=0.000l). When the minerals were used alone,

both copper and the mineral mix reduced disease severity from 3 to approximately 2.4

(Fig. 1) and increased plant weight from 163 to approximately 217 mg (Fig. 2)

(P<0.0002). In contrast, ammonium-molybdate and zinc amendments resulted in slight

but not significant increases in disease severity and reductions in plant fresh weight.

Combinations of CHAO with any of the mineral treatments significantly reduced

disease (Fig. 1)(P=D.0001) and combinations with copper and the mineral mix

significantly increased plant fresh weight (Fig. 2)(P<0.00()4). Ammonium-molybdate

had no effect on disease rating or plant weight when used in combination with CHAO.

The biocontrol activity of CHAO, expressed as the sum of the FORL treatment minus

the CHAO plus FORL treatment, was improved over 50% by copper and zinc

(P=0.0241) (Fig. 1). The ability of CHAO to improve plant growth was increased four¬

fold by zinc. Biocontrol activity was not enhanced by ammonium-molybdate nor the

mineral mix.

In the absence of FORL inoculation, small lesions developed on a few plants,

possibly from aerial dissemination from sporulating stem lesions in treatments with

FORL. There was no significant difference m chance infection between the absolute

control (no FORL, no CHAO, and no minerals, rating 0.03) and any of the controls with

80

minerals (ratings ranging from 0.01 for the mineral mix to 0.06 for the ammonium-

molybdate). In the absence of pathogen and biocontrol agent, none of the minerals, at

the concentrations used in this study, had a significant effect on plant growth (Fig. 2).

Higher concentrations (66 and 100 ug/ml) of ammonium-molybdate and the mineral

mix did have slight phytotoxic effects on tomato growth in preliminary tests (data not

shown).

35 -

to 2 5-

S 15-

1

0 6

0

1

35

3

S 2 5-

Q) 15

0 5

Q

O)

FORL alone

SHHrF

fill«56

FORL plus CHAO

a_

Mam.

me.

m

1 4

c 12-o

t> 1-

1 08-

to

2°4

Û 0 2

0

Biocontroi activity

ab

ïëPPtÉ

füg

be be

None Zn Cu Mix

Mineral amendment

NH -Mo4

Figure 1. Effect of mineral amendments

on tomato crown and root rot disease

seventy caused by Fusarium oxysporum

f.sp. radicis-lxcopersici (FORL alone)

and on biocontrol using Pseudomonas

fluorescens (FORL plus CHAO). One¬

time mineral amendments to nutrient

solution at 33 ug/ml included Zn or Cu

(as EDTA complexes), ammonium

molybdate (NHrMo), or a mix of 1/3

each. Control plants (none) received

only nutrient solution. Tomato plants

were grown in soilless rockwool culture

infested with FORL for two weeks and

evaluated for disease severity on a scale

of 0 to 4 w here 0=symptomless and

4=dead or nearly so. The biocontrol activity of CHAO was expressed as the product of (FORL

alone) - (FORL plus CHAO) = disease reduction. Values represent the means of eight

replications. Bars with the same letter are not significantly different according to Fisher2s

protected (P=0.05) LSD test. LSD values were 0.28 (FORL alone), 0.33 (FORL plus CHAO),

and 0.44 (Biocontrol acthitv).

Effect of minerals on pathogen growth, conidia production, and fusaric acid

production. Minerals had a differential effect on the pathogen. Zinc, copper, and the

mineral mix increased total fungal biomass (P < 0.007; R2= 0.42, 0.57, 0.32,

81

respectively) (Fig. 3A) and the number of conidia (P < 0.002; R2= 0.23, 0.44, 0.31,

respectively) (Fig. 3B). Fusaric acid production was almost completely repressed by

zinc, copper, and the mineral mix at concentrations as low as 10 (lg/ml (P < 0.001; R2 =

0.35. 0.57, 0.36, respectively) (Fig. 3C). In contrast, ammonium-molybdate did not have

a significant effect on conidia production, total fungal biomass, or FA production (Fig.

3). Recovery of FA produced by FORL or synthetic

A

sz

as

szen

TO

a.

400

300

200

100-

0-

400 '

300

200

100 -\

Iffli

«pisjgegg

Si®

iff

ab

- be

I

B

Figure 2. Effect of mineral amendments on

fresh weight of tomato plants A, grown in

nutrient solution without pathogen or

biocontrol agent; B, challenged with FORL

alone (no biocontrol agent); and C,

protected with Pseudomonas fluorescens

strain CHAO (FORL plus CHAO). See Fig.

1 for the experimental procedure. In the

absence of the pathogen A, mineral

amendments had no effect on plant fresh

weight and means were not compared

using Fisher2s protected LSD test. For

treatments with the pathogen, LSD values

were B, 42.7 and C, 44.3.

lineral amendment

FA was not affected by zinc nor ammonium-molybdate at the highest mineral

concentration tested (100 jig/ml) compared with treatments without minerals, but

recovery of this phytotoxin was reduced 5 to 10% by copper amendment.

Fiffect of fusaric acid on CHAO secondary metabolism. Synthetic FA at

concentrations of 0.37 (ig/ml or greater completely repressed production of both FHL

(P=0.02, R2=0.87) and monoacetylphloroglucinol (P=0.09, R:=0.65) antibiotics by

82

CHAO in PCG medium (Fig. 4). Fusaric acid at 100 pg/ml was sufficient to completely

repress PHL production by the antibiotic-overproducing derivative CHA0/pME3424

--Zri-S-Cu"À-NH4-Mô0-Mi

£4000

a. 3000

T32000

O

o

Ü 1000o

50

"55 250S-

..-< 200,.c

ra

a) 150£

>< 100-a

CO 50en

cn 0LL.

2000

.1.:,

T) O) 1600CD

03 S1200

O r"

CO 33800

~) r~

U_O) 400

0--

IX

Figure 3. Effect of mineral amendments

2 (0 to 100 pg/ml zinc and copper as

„^.--fl EDTA complexes, ammonium

molybdate, and a mix of 1/3 each) to

Czapek-Dox broth on fungal

microconidia production (A), growth

(B), and fusaric acid production (C).

FORL was incubated for 8 days (24 °C,

120 rpm). Fusaric acid was quantified

with HPLC and expressed relative to

biomass (mycelia and microconidia).

Vertical bars indicate standard error of

the mean.

20 40 60 80 100

Mineral amendment (ug/ml)

which produced six-times more PHL than the wild-type in the absence of FA (Table 1).

Production of pyoluteorin antibiotic by CHAO in GNB medium was reduced

approximately 50% by 100 pg/ml FA (Table 1). Fusaric acid did not affect the

production of other secondary metabolites which are regulated by gacA and apdA genes,

including hydrogen cyanide and extracellular proteases (Table 1). Fusaric acid reduced

the production of pyochelin siderophore but increased slightly the production of

salicylic acid, a precursor for pyochelin (Table 1 ), Inhibition of antibiotics and

pyochelin by FA was neither reversed nor reduced with additon of Zn2*" at 66 itg/ml

(Table I).

83

160

"D

1?0->>„Ü O -

*+—» "*

lô.E80-

CD

40

-a- PHL

-A—mPHL

0.1 0.2 0.3

Fusaric acid (ug/ml)

-rffl-0.4

Figure 4. Effect of fusaric acid on production of the antibiotics monoacetylphloroglucinol

(mPHL) and 2,4-diacetylphloroglucinol (PHL) b\ Pseudomonas fluorescens. Biocontrol strain

CHAO was grown for 48 h in PCG broth amended with synthetic fusaric acid (0 to 200 ug/ml)

and antibiotic production was quantified with HPLC after ethy lacetate extraction. At

concentrations above 0.37 ug/ml. no antibiotics were detected and data were not included in the

analysis. Data were log transformed prior to analysis. Values represent the means (+ standard

error of the mean) of six extractions per concentration.

Fusaric acid at 200 pg/ml or less had no effect on the pH of PCG or GNB media

at the start or end of the experiment (approximately pH 6.5 and 8.1, respectively), nor

was bacterial growth affected (5 to 8x10' CFU/ml in treatments with and without FA).

Growth of CHAO resulted in neither degradation nor interference with the recovery of

FA from either PCG or GNB medium (60% recovery with or without CHAO after 2

days incubation). Likewise, addition of synthetic fusaric acid to 48 h PCG cultures of

CHAO had no effect on the recovery of preformed PHL. The antibiotic was recovered at

(pg/ml ± standard error of the mean). 0.75 ± 0.2 without FA, 0.79 ± O.lwith 100 p_g/ml

FA, and 0.76 ± 0.2 with 500 pg/ml FA,

Effect of zinc amendments on in situ metabolite production by the pathogen

and biocontrol agent. Extracts from the rockwool assay contained compounds that

comigrated with and had ultra-violet spectra identical to FA and PHL reference

compounds. In the absence of zinc, FA was detected in FORL and FORL plus CHAO

84

TABLE

1.Influenceoffusaricacid(FA)

onse

cond

arymetaboliteproduction

byCHAO.

Yield(ng/ml/108

CFU)'

Protease'

Amendment'

PHL

PLT

SAL

PCH

(units/lOcf

P.fluorescensCHAO

None

58.8±3.2

15.6±

4.0

13.32

1.2

819.62

74.2

10.92

0.5

FA

<0.01

8.3+

1.3

24.9±

2.5

513.1±

61.1

9.8±0.3

FA

plus

zinc

<0.01

9.2±

1.7

22.7±

1.4

520.42

138.9

nd

Zinc

87.8+

6.2

32.7±

10.4

18.32

4.5

962.6±297.9

nd

HCN'

P.fluorescensCHA0/piML3424

None

303.9214.3

FA

<0.0I

nd

nd

nd

nd

nd

nd

nd

nd

+ + + +

"

Autoclavedmediawasamendedwithfusaricacid

togive

100ug

/nil

andwith66ug/ml

Zn2r

added

asZnSO.x7H,G.

'Hydrogencyanide(HCN)wasestimatedusingindicatorpaper,extracellularproteasewasquanti

fied

usingtheazocascin

reac

tion

,and

2,4-

diac

etyi

phloroglucinol

(PHL),pyoluteorin(PLT),

salicylicacid(SAL),andpyochelin(PCH)werequantified

usingHPLC

anal

ysis

asdescribed

in

MaterialsandMethods.Proteaseandpyoluteorinweredetermined

inGNB

medium.Othermetabolitesweredetermined

inPCGmedium.Values

representthemean

ofsixre

plic

atebrothcultures(±standarderrorofthemean).nd=notdetermined.

85

TABLE

2.In

situpr

oduc

tion

offusaricacid(FA)by

F.

o.f.

sp.ra

dici

s-ly

cope

rsic

i(FORL)and2,4-diacetylphloroglucinol

(PHL)by

P.fluorescens

CHAO

inthetomatorockwoolbi

oassay

withandwithout33ug/mlZn

2+asZn-EDTA.

Microorganismsadded

Zn-EDTA

PHL

(ugper

plant)y

FA

(ugperpl

ant)

CHAO

GO5CFU/ml)

FORL

(104CFU/ml)'

None

FORL

alone

CHAO

alone

FORL

plus

ClIAO

<0.07

<0.45

00

<0.07

<0.45

00

<0.07

5.1

(2.5

to7.4)

010.9(22.

3)<0.07

0.4(0

to0.

71)

09.1(±1.7)

0.29(0.11

to0.

41)

<0.45

7.9(±

1.9)

0

0.39(0.12to0.

65)

<0.45

6.3(r

: 2.1)

0

<0.07

5.7

(2.3

to7.9)

5.5(r

i 3.6

)9.7(±

1.9)

0.48(0

.26to0.

66)

<0.45

9.5(+

: 2.9)

5.6(±1.8)

'"

Values

arethemeans

ofthreeextractionswithrangesgiven

inpa

rent

hesi

s.Each

extractionconsistedoftwo

tray

sof42tomato

plan

tsgrown

fortwo

weeks

inloekwool.

'

Microbialnumbers

arethemeans

forthree

tria

lswith±

standarderrorofthemeans

given

inpa

rent

hesi

s.

86

treatments at approximately 5 u.g per plant (Table 2). In the presence of zinc, the

Phytotoxin was not detected in two of three trials when FORL was inoculated alone and

it was never detected when FORL was coinoculated with CHAO. In the absence of zinc,

PHL was detected in the CHAO treatment at approximately 0.3 jug per plant but it was

not detected when CHAO was coinoculated with FORL. When zinc was added to the

nutrient solution, approximately 0.4 pg per plant were detected when CHAO was tested

alone, and 0.5 u.g per plant were detected when CHAO was coinoculated with FORL.

This indicates that zinc had no direct effect on antibiotic production by the biocontrol

agent but rather it reduced fusaric acid production by the pathogen, thereby creating an

environment conducive for normal levels of PHL biosynthesis. Neither PHL nor FA

were detected in extracts of the control treatments.

Zinc amendment had no effect on the number of CHAO"1 CFU when the

biocontrol agent was tested alone, but it slightly increased the number of CHAO'" CFU

when the biocontrol agent was coinoculated with FORL (Table 2). In contrast, while

zinc had no effect on the number of FORL CFU when the pathogen was tested alone,

zinc reduced the number of FORL CFU when the pathogen was coinoculated with

CHAO'". This suggests that zinc had a positive effect on the competitiveness of

CHAO'" and a negative effect on the competitiveness of FORL.

Discussion

Pseudomonas fluorescens CHAO has a broad-spectrum biocontrol activity against

diseases caused by soilborne fungal pathogens (Voisard et al. 1994). Against Fusarium

crown and root rot of tomato, however, CHAO was only moderately effective. In our

screening program using this disease assay to identify new biocontrol strains, CHAO

would probably not have been carried forward to the next step in the screening process.

While selection of new strains that may be especially suited for particular pathosystems

and environments is certainly worthwhile, there are also incentives for improving the

activity of an otherwise good strain.

Application of strain mixtures (Duffy et al. 1996, Voisard et al. 1994) and

genetic enhancement of antibiotic biosynthesis (Schnider et al. 1995. Weller and

Thomashow 1994) are two approaches that have been reported to improve the efficacy

of CFIAO and other biocontrol strains, "faking a new approach, we investigated the use

87

of mineral amendments as a way to create a more favorable environment for biocontrol

to occur. This idea is based on the managment of certain diseases with mineral

fertilization regimes. In most cases, the minerals work by directly reducing pathogenic

activity and/or improving host tolerance (Engelhard 1989, van Broembsen and Deacon

1997). For example, increasing the ratio of nitrate to ammonium forms of nitrogen

reduces Fusarium wilt of tomato because the use of nitrate-nitrogen raises soil pFI,

reduces pathogen reproduction and propagule germination, and reduces the sensitivity

of tomato to pathogen Phytotoxins, particularly fusaric acid, while ammonium forms

have the opposite effect (Barna et al. 1983, Jones et al. 1989). Mandai and Sinha (1992)

have suggested that zinc and other minerals reduce tomato wilt by inducing host

resistance. There are a few cases though where minerals appear to reduce disease by

exerting an indirect beneficial effect on indigenous and introduced antagonistic

microorganisms (Elmer 1995, Huber 1989). Zinc soil content has been found to be

positively correlated with the biocontrol activity of introduced P. fluorescens l-l1^

(Weiler and Thomashow 1994). Calcium has both a direct negative impact on the

activity of postharvest pathogens of apple and citrus and an indirect positive impact on

the biocontrol activity of saprophytic yeasts (Droby et al. 1997, McLaughlin et al.

1990).

We found that copper and zinc significantly improved the biocontrol activity of

CHAO against FORL in soilless tomato culture. Copper reduced disease resulting in

improved plant growth when it was used alone, and it had an additive effect when used

in combination with CHAO. In contrast, zinc had no direct effect on disease when used

alone indicating that this mineral indirectly reduced disease through some influence on

the interaction between the biocontrol agent and the pathogen. Initially, we thought that

zinc improved biocontrol by stimulating the biosynthesis of bacterial antibiotics

important in disease suppression. Zinc and other trace minerals stimulate the in vitro

production of 2,4-diacetylphloroglucinol. pyoluteorin. and phenazine type antibiotics

(Duffy and Défago 1996. Slininger and Jackson 1992), and stabilize regulatory genes

critical for antibiotic production in fluorescent pseudomonads (Duffy and Défago 1995).

However, zinc amendment resulted in only a slight increase in the in situ production of

2,4-diacetylphloroglucinol in situ, from approximately 0.3 to 0.4 pg per plant, and did

not affect bacterial growth.

88

Mineral nutrition is also important in the biology of the pathogen. We found that

zinc and copper increased total biomass, increased conidiation, and altered the profile of

secondary metabolites produced by FORL. The tomato crown and root rot pathogen

produced fusaric acid, a pyridine-carboxylic acid (chemically, 5-butylpicolinic acid)

with nonspecific phytotoxic activity that contributes to wilt and rot diseases of various

crops caused by F. oxysporum (Chakrabarti and Basu Chaudhary 1980, Kern 1972,

Remotti and Löffler 1996, Toyoda et al. 1988). Fusaric acid production was completely

repressed by zinc and copper at concentrations as low as 10 ug/ml and by a mineral mix

with 3 p,g/ml of each of these. Ammonium-molybdate, which had no effect on disease

nor on biocontrol. also did not affect growth and fusaric acid production by FORL, even

at a concentration of 100 pg/ml. Fusaric acid production was not detected in the tomato

bioassay in two trials and at low concentration (0.7 jig per plant) in a third trial when

plants were treated with zinc at 33 u,g/ml, but it was detected at 5.1 u,g per plant in the

absence of zinc. Depending on concentration, zinc has been reported to increase (3 uM)

or decrease (6 uM) fusaric acid production by the tomato wilt pathogen, F. oxysporum

f.sp. lycopersici (Egli 1969). Fusaric acid production by nonpathogenic and moderately

pathogenic strains tended to be more sensitive to zinc repression compared with

production by highly pathogenic strains (Chakrabarti and Basu Chaudhary 1980, Kern

1972). Zinc at 15 to 150 pM increased production of aflatoxin by Aspergillus

parasiticus but at 150 uM or more toxin production was repressed (Weinberg 1977).

The fact that in our experiment disease severity was not reduced when fusaric acid

production was repressed by zinc amendment does not preclude a role for this

Phytotoxin in disease development under other conditions. Rather, this suggests that

FORL produces additional metabolites (e.g.. other phytotoxms and lytic enzymes)

which may have contributed to disease development. One or more of these

pathogenicity factors may have been insensitive to repression by zinc or may even have

been stimulated by zinc.

Zinc clearly had an effect on fusaric acid production by the pathogen and on

antibiotic production by the biocontrol agent. However, neither interaction alone seemed

to explain the influence of zinc on biocontrol. A simple experiment intended to show

that CHAO did not degrade fusaric acid, unexpectedly revealed that fusaric acid at

89

concentrations as low as 0.1 u.g/ml repressed production of 2,4-diacetylphloroglucinol

by the biocontrol agent. This suggested that zinc may indirectly improve biocontrol by

removing fusaric acid from the system, thereby creating an environment more

conducive to antibiotic production by the bacterium. Indeed, when microbial metabolite

production was measured in the tomato bioassay, zinc amendments repressed fusaric

acid production in situ and this was accompanied by increased production of 2,4-

diacetylphloroglucinol by CHAO in situ. This is perhaps the first evidence that

pathogens can have a direct impact on the efficacy of biocontrol agents at a mechanism

level. Previously, fungal pathogens have been shown to have a selective influence,

positive and negative, on the proliferation of bacterial biocontrol agents in the

rhizosphere of wheat (Mazzola and Cook 1991) and to influence bacterial Chemotaxis

(Arora 1986).

The mechanism by which fusaric acid represses antibiotic production by CHAO

is uncertain. Fusaric acid is toxic to animal, plant, fungal, and bacterial cells primarily

because it interferes with cell function (eg., respiration, membrane integrity, ATP levels,

and enzyme activity) (Fernandez-Pol et al. 1993, Kern 1972, Porter et al. 1996,

Prabhakaran et al. 1983). Growth of CHAO was not reduced even at fusaric acid

concentrations well above that needed for repression of antibiotic production. Media

pH, which is important in antibiotic biosynthesis (Duffy and Défago 1996, Slininger and

Shea-Wilbur 1995), was also not affected by fusaric acid. Fusaric acid did not interfere

with the recovery of preformed antibiotic, a further indication that some step in the

biosynthetic pathway was interrupted. Production of hydrogen cyanide and extracellular

protease was not repressed suggesting that the target site for fusaric acid is downstream

from the global regulatory genes gacA and apdA, perhaps at the promoter or

transcription level of the polyketide antibiotics 2,4-diacetylphloroglucinol and

pyoluteorin. Competition with enzymes involved in the biosynthesis of these antibiotics

may also play a role (Diringer et al. 1982. Kern 1972, Pandy et al. 198 l, Prabhakaran et

al. 1983), but this would have to be highly effective considering that a small amount of

fusaric acid is sufficient to inhibit antibiotic production even by an antibiotic-

overproducing derivative of CHAO.

Chelation is a factor in the toxicity and antidepressant activity of fusaric acid

(Bochner et al. 1980. Kern 1972). The role of chelation in antibiotic repression is

90

difficult to confirm because this would affect fusaric acid availability, thereby

precluding observation of possible specific interactions with the cell (Kern 1972). While

zinc amendments did not relieve fusaric acid-mediated antibiotic repression, repression

may have resulted from chelation of iron or other minerals that directly or indirectly

influence antibiotic biosynthesis and for which fusaric acid has a higher affinity. The

fact that hydrogen cyanide, which is sensitive to iron availability (Voisard et al. 1994),

was not repressed by fusaric acid and that antibiotic production was not affected by the

high-affinity iron chelator EDDHA [(ethylenediamine-di(o-hydroxyphnylacetic acid)]

(B. Duffy, unpublished data) suggests that chelation was not the only factor in fusaric

acid-mediated repression of antibiotic production by CHAO.

Trace mineral amendments are an inexpensive way to improve the biocontrol

activity of certain bacterial strains. Formulations that efficiently supply minerals to the

target strain may further improve their availability and effect on biocontrol which means

lower closes and reduced costs. Selectively providing the minerals to the biocontrol

strain should limit potential nontarget toxicity to other beneficial microorganisms

(Babich and Stotzky 1978, Page et al. 1996) and avoid increases in pathogenic activity

(Jones et al. 1989). Identification of mineral amendments that favor biocontrol may also

provide clues to soil factors or components of nutrient solutions in hydroponic culture

that will improve the level and reliability of biocontrol. Zinc amendments improved the

biocontrol activity of P. fluorescens 2-79, and the variable performance of this strain in

the field was associated with soil concentrations of DTPA-extractable zinc (Weiler and

Thomashow 1994). Identification of the fungal pathogenicity factor, fusaric acid, as a

negative signal in the biocontrol activity of CHAO may explain in part the moderate

performance of this strain against Fusarium crown and root rot of tomato. Fusaric acid is

produced by a wide range of plant associated fusaria (Abbas et al. 1989, Bacon et al.

1996) and it may have an influence on biocontrol in other pathosystems. When faced

with fusaric acid producing pathogens, better disease control might be achieved by

using biocontrol strains insensitive to fusaric acid-mediated antibiotic inhibition, or by

using strains capable of degrading fusaric acid (Toyoda et al. 1988).

91

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94

Voisard, C, Bull, C. T., Keel, C, Laville, L, Maurhofer, M., Schnider, U., Défago, G., and

Haas, D. 1994. Biocontrol of root diseases by Pseudomonas fluorescens CHAO: current

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95

Chapter 4

A Fusarium Pathogenicity Factor Blocks Antibiotic Biosynthesis by

Antagonistic Pseudomonads

Abstract

Fusaric acid, a non-specific pyridine-carboxylic acid phytotoxin produced by Fusarium

oxysporum, was identified as a negative signal in biocontrol of Fusarium crown and root rot of

tomato. Fusaric acid (100 ug/ml) repressed production of the antibiotic 2,4-

diacetylphloroglucinol (PHL) by some but not all biocontrol strains of Pseudomonas

fluorescens. Strains sensitive to fusaric acid repression were significantly less effective

biocontrol agents. Production of PHL was a primary mechanism of action for strain Q2-87

which was not sensitive to fusaric acid; whereas, PHL had no role in CHAO which is sensitive to

fusaric acid. The residual level of control with this strain was attributable in part to production

of the biocide hydrogen cyanide (HCN).

Published as IOBC wprs Bull. 21(9):145-148.

Introduction

Fusarium oxysporum fsp. radicis-lycopersici causes crown and root rot of tomato, an

increasingly important disease in hydroponics production systems. Fluorescent

pseudomonads have been effective for biocontrol of this and other Fusarium diseases.

However, certain biocontrol strains tend to perform better than others. Our objective

was to determine the molecular basis behind this with the long-term aim of improving

strain selection procedures.

Materials and Methods

We screened an ecologically and genetically diverse collection of 42 Pseudomonas

fluorescens strains for biocontrol of Fusarium crown and root rot of tomato. All bacteria

carry a conserved phlD gene essential for biosynthesis of the antibiotic, PHL. In

addition, all strains produce HCN. Keel et al. (1996) characterized these strains into

three distinct groups based on amplified ribosomal DNA restriction analysis (ARDRA),

96

In this study, we compared the strains for biocontrol activity against crown and

root rot in a soilless rockwool system as described by (Duffy and Défago 1997). Tomato

seeds (Lycopersicum esculentum cv Bonnie Best) were placed in rockwool blocks in

shallow trays, and the rockwool was saturated with a mineral nutrient solution. The

nutrient solution was inoculated with both the pathogen (106 microconidia plus mycelial

fragements per ml) and the bacterium (10 CFU per ml). After 2 weeks growth in the

greenhouse, plants were removed from the rockwool and disease was evaluated using a

scale of increasing severity from 0 to 4.

Then, we determined the influence of fusaric acid, a pathogen phytotoxin, on

bacterial antibiotic production. Bacteria were grown in liquid PCG medium (Duffy and

Défago 1997) with 100 |ig/ml fusaric acid (Sigma Chemical Co.. St. Louis. MO, USA).

PHL production and fusaric acid degradation was quantified after 48 hours using HPLC

as previously described (Duffy and Défago 1997). The minimum-concentration of

fusaric acid needed to repress PHL production was determined with strain CHAO.

CHAO was grown 48 h in PCG medium amended with fusaric acid at 0-200 u.g/ml and

PHL production was quantified as above. The relationship between biocontrol and

sensitivity to fusaric acid was evaluated using SAS regression procedures (SAS

Institute, Cary, NC. USA). Disease severity rating was plotted against the amount of

PHL produced in the presence of fusaric acid.

Finally, the relative role of PHL and HCN in biocontrol was determined with

metabolite-negative insertion mutants and restored mutants of CHAO and Q2-87

(Vincent et al. 1991, Voisard et al. 1989). Bacteria were compared with the wild-type

strains in the rockwool assay as described above. Pathogen sensitivity to PHL was

determined after 7 days growth on 2% malt extract agar amended with 0-150 Ug/ml

synthetic antibiotic following Keel et al. (1992).

Results and Discussion

An ecologically and genetically diverse collection of 42 Pseudomonas fluorescens

biocontrol strains was characterized into three groups using ARDRA analysis (Keel et

al. 1996). There was a wide variation between strains m the level of biocontrol provided

against Fusarium crown and root rot of tomato in a rockwool soilless system. This

variation was attributed to differential responses of the strains to fusaric acid, a pathogen

97

Phytotoxin. Fusaric acid repressed production of PHL by all ARDRA 1 strains but did

not affect bacterial growth. For example, antibiotic production by strain CHAO was

completely repressed with 0.12 jig/ml fusaric acid (Fig 1) and concentrations as high as

200 pig/ml did not reduce total CFU (data not shown). Strains in ARDRA 2 and 3

groups were less sensitive or resistant to fusaric acid repression. Regression analysis

demonstrated a significant inverse relationship between sensitivity to fusaric acid in

vitro and biocontrol activity (data not shown). In other words, sensitive strains were

inferior whereas resistant strains were superior biocontrol agents.

PHL

01 02 03

Fusaric acid (ug/ml)

t^—mPHL Figure 1. Fusaric acid repression of

2,4-diacetylphloroglucinol (PHL) and

the precursor antibiotic mono-

acetylphloroglucinol (mPHL) in P.

fluorescens CHAO after 48 h in PCG

liquid medium amended with fusaric

acid (0-200 pg/ml). At concentrations

04

above 0.37 pg/ml, no antibiotics were detected using HPLC. (From Duffy and Défago 1997)

Fusarium oxysporum was sensitive to pure PHL (IDM) between 30 and 50 |ig/ml).

This suggests that PHL could confer a competitive advantage to pseudomonads over the

pathogen. Indeed, PHL production is a primary mechanism of biocontrol in fusaric-acid

resistant strains. Genetic interuption of PHL biosynthesis genes in Q2-87 substantially

reduced biocontrol activity (Table 1). In contrast, PHL had little if any role in biocontrol

in the fusaric-acid sensitive strain CFIAO. In this strain, HCN production contributed to

the moderate level of biocontrol. This highlights the advantage of having multiple

biocontrol mechanisms on hand to cope with different environmental conditions.

Our findings have practical applications for strain selection. Raaijmakers et al.

( 1998) recently outlined a screening procedure for biocontrol pseudomonads based on

selection of strains carrying conserved PHL biosynthesis genes. We suggest that the

process can be further refined by selecting strains capable of expressing these genes in

98

TABLE 1. Relative role of 2,4-diacetylphloroglucinol (PHL) and hydrogen cyanide (HCN) in

biocontrol of Fusarium crown and root rot by Pseudomonasfluorescens

Strain Phenotype Disease rating Plant fresh weight

(0-4) (mg)

None —- 2.80(0.16) 130.6 (2.8)

CHAOwt PHL+, HCN+ 2.41(0.10) 170.2(12.6)CHA630 PHL minus, HCN+ 2.53(0.05) 160.2(11.1)

CHA630/pMON5ll8 PHL restored, HCN+ 2.33(0.07) 165.3(7.3)CHA5 PHL+, HCN minus 2.65(0.13) 142.2(11.4)

CHA5/pME3013 PHL+, HCN restored 2.42(0.06) 175.4(13.0)

Q2-87 wt PHL+HCN+ 1.41(0.11) 239.2(7.8)

Q2-87::Tn5-1 PHL minus, HCN+ 2.48(0.11) 162.6(13.0)

Q2-87::Tii5-l/pMON5ll8 PHL restored, HCN+ 1.52(0.10) 218.3 (6.7)

Values (± SE) represent the means of 5-6 replications with 12 plants each. All treatments were

challenged with F. oxysporum fsp radicis-lycopersici and evaluated after 2 weeks.

particular environments. In the case of strain selection for biocontrol of Fusarium

diseases, it is clearly important to select not only for PHL producing strains but for PHL

strains with the capacity to produce this antibiotic in the presence of the pathogen. We

further observed that biocontrol was not related to the amount of PHL produced in the

absence of fusaric acid. This strengthens the argument that screening based on in vitro

production of antimicrobial compounds should be evaluated qualitatively, rather than

quantitatively. Strains that produce low levels in vitro in a particular medium, may

produce much more under other conditions where it may be more important for

biocontrol. Indeed, PHL production by the strains used in our study differed greatly

when evaluated in different media (PCG liquid in this study. KMB and malt extract agar

in Keel et al. 1996).

This study also shows that even though biosynthetic genes may be conserved

among strains, the regulation of these genes can differ dramatically. Continuing efforts

to identify these differences will shed new light on antibiotic regulation. Ultimately, this

may lead to novel approaches for improving bacterial interactions with the

environmental conditions tinder which they must operate.

99

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fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions. EMBO

.18:351-358.

Qpiifp f Apr / I

Blank feaf I

101

Chapter 5

Macro- and Microelement Fertilizers Influence the Severity of Fusarium

Crown and Root Rot of Tomato in a Soilless Production System

Abstract

Host nutritional variables were evaluated to reduce the severity of crown and root rot of tomato

caused by Fusarium oxysporum f. sp. radicis-lycopersici. Tomato 'Bonnie Best' seedlings were

grown in a pathogen-infested soilless rockwool system in the greenhouse, and were fertilized

with a nutrient solution that was amended with macro- and microelements at various rates.

Disease was evaluated after two weeks using an index of 0 to 4 and plant fresh weight was

measured. Regression analysis indicated that disease severity was significantly increased by

ammonium-nitrogen [NHC1, (NH4)6Mo70,4. and (NHASOJ, NaH2P04- H,0, Fe-EDDHA,

MnSO,, MoO,, and ZnSO, • 7H,0. Disease was reduced by nitrate-nitrogen [Ca(N03), • 4H,0],

and CuSO, • H,0. Low rates of NH4NO, (39 to 79 mg N /liter) reduced disease, but this effect

was reversed as rates increased above f 00 mg N /liter. Disease was not affected by MgSOt

7H,0. In all cases, plant growth was inversely related to disease severity. Mineral fertilizers

had no effect on nutrient solution pH. This information sheds new light on environmental

factors that influence plant-pathogen interactions, and may be applied to develop a

management strategy for Fusarium crown and root rot based on host nutrition.

HortScience, in press.

Crown and root rot of tomato (Lycopersicum esculentum Mill.) (also referred to as foot

and root rot) caused by Fusarium oxysporum Schlechtend.:Fr. f.sp. radicis-lycopersici

Jarvis & Shoemaker was first described in Japan in the late 1970s, and has since become

an economically important problem in greenhouse tomato production world-wide (e.g.,

throughout Europe, North America. Japan, and Israel) (Jarvis. 1988). It is considered the

most destructive tomato disease caused by a non-zoosporic pathogen in soilless

hydroponics production systems where reductions in marketable yield can exceed 60 %

102

(Mihuta-Grimm et al., 1990; Stangheilini and Rasmussen, 1994). Tomato plants can be

infected at any time, but losses are especially severe when infection occurs at the

seedling stage. Symptoms include dark-brown necrotic lesions that form on the roots

and crown region and may extend up the hypocotyl, stem and foliage, localized

discoloration of the vascular tissues, followed by chlorosis, wilting and death. The

pathogen is spread via infected transplants, which often carry latent infections. It can

then grow to some extent through soil to infect adjacent plants (Hartman and Fletcher,

1991; Louter and Edgington, 1990). The threat to tomato production is further increased

by infection from airborne conidia of 72 oxysporum f.sp. radicis-lycopersici that arc

produced in stem lesions and are dispersed by wind and fungus gnats [Bradysia spp.

(Diptera: Sciaridae)] (Gillespie and Menzies. 1993; Rowe and Farley, 1981).

Control of Fusarium crown and root rot on tomato has been difficult. No highly

resistant and commercially acceptable cultivars are available (Rowe and Farley, 1981).

Fungicides, including benomyl, captafol, imazalil, thiram, and prochloraz-Mn, provide

inconsistent control, leave problematic residues in edible tissues, and are often

phytotoxic even when applied at recommended rates, especially on seedlings (Hartman

and Fletcher, 1991; Jarvis. 1988, 1992; Mihuta-Grimm et al., 1990). Biological control

utilizing lungal (i.e., Frichoderma harzicmunu non-pathogenic Fusarium oxysporum and

F. solanf) and bacterial agents (i.e.. Bacillus suhtilis and Pseudomonas spp.) typically

provides only a moderate level of disease suppression in the greenhouse and field

(Bochow et al., 1996; Duffy and Défago. 1997; Hartman and Fletcher. 1991; Louter and

Edgington, 1990; M'Piga et al., 1997; Sivan et al., 1987). Allelopathy from lettuce and

dandelion residues incorporated into soil provides limited control (Jarvis, 1992; Jarvis

and Thorpe, 1981) but is technically impractical in hydroponics production systems.

103

Alternative control measures are needed, ideally with the aim of developing an

integrated disease management strategy.

It is well documented that the nutritional status of a plant has a major impact on

disease susceptibility, and this has be exploited for suppressing a variety of diseases

(Engelhard, 1989). Notable examples are Fusarium wilts caused by F. oxysporum

formae speciali, with reports dating to the 1920s that describe the beneficial effect of

lime amendments (Jones et al., 1989). Since then, the effects of most major and minor

nutrients on wilt diseases have been studied. Jones and co-workers (Jones et al. 1989)

applied this information to develop an effective fertilizer-based management strategy for

Fusarium wilt of tomato caused by 72 oxysporum f. sp. lycopersici which is used in

commercial production systems in the USA. Similar approaches have been successful

for control of Fusarium wilts of other vegetable and ornamental crops (Elmer, 1992;

Jones et al., 1989; Schneider, 1985).

In contrast, little is currently known about the influence of plant nutrition on

crown and root rot of tomato. Previous studies tested single concentrations of

nitrogenous fertilizers and sodium chloride (Jarvis and Thorpe, 1980; Woltz et al..

1992), and did not examine the influence of other potentially critical mineral

amendments, particularly microelements. The objective of our study was to compare the

effects of several macro- and microelements (various ammonium-N sources, nitrate-N,

sodium phosphate, iron, molybdenum, and copper-, magnesium-, manganese- and zinc-

sulphates) on disease severity and growth of tomato seedlings. Minerals were tested

across a range of added concentrations to provide more information that can be applied

to develop a fertilizer-based management strategy and for integrating mineral

104

amendments with other control approaches (e.g., biocontrol) which may be sensitive to

mineral levels.

Materials and Methods

The influence of various mineral amendments on Fusarium crown and root rot was

evaluated in a non-circulating hydroponics system. Pregerminated [2—3 days on 0.85%

water agar (Oxoid®, Hampshire, England) at 24 °C; 2—4 mm long primary root]

tomato seeds cv. Bonnie Best were planted on rockwool cubes (3.5 cm2 diameter x 4 cm

deep; one seed per cube; Grodania A/S, Hedehusene, Denmark) in square plastic trays

(5.5 cm deep). The rockwool was saturated with 800 ml of dilute (1/4 strength) Knop

nutrient solution (Ziegler, 1983) containing (mg/liter): Ca(NO,), 4H,0, (250); KH2POt,

KCl, and MgSOr 7H.O, (each 62.5); and Fe-EDDHA Lethylenediamine-di(o-

hydroxyphenyl-acetic acid); Sequestrene 138 Fe, Novartis AG, Basel, Switzerland], (5).

Autoclave-sterilized stock solutions of the various minerals tested [Ca(NO,);4H20,

CuS04-5H20, MgSO,-7ILO. MnS04, MoO„ NaH,PO,H20. NH4C1, (NH,)6Mo7Oit-4H,0.

NH,NO,, (NHtLSO(, ZnSO,-7H,0] were added to the nutrient solution to give a range of

final concentrations (see Figs. 1—4) and the pH was measured with a Digital-meter

(Auer Bittmann Soulié AG, Zürich, Switzerland). Prior to saturating the rockwool, the

nutrient solution was inoculated with F. oxysporum f.sp. radicis-lycopersici to give

approximately 10s microconidia plus mycelial fragments per ml. as previously described

(Duffy and Défago. 1997). Mycelial fragments were included because in preliminary

experiments, inoculation with microconidia alone resulted in little or no disease.

Inoculum was produced by growing the pathogen in 150 ml of 222 malt extract broth

(pH 5.5; Oxoid®) in 500 ml baffled Erlenmeyer flasks at 24 °C for 5—7 days with

105

shaking at 130 rpm. Cultures were centrifuged ( 15 min at 2 200 g) to collect fungal

biomass which was then briefly homogenized in an electric blender immediately before

adding to the nutrient solution.

Plants were grown in a growth chamber in the greenhouse with 16 h light: 8 h

darkness, 22 °C 'day2 18 °C 'night', and 70 c,c RH. Lower temperatures are favorable

for development of crown and root rot, in contrast to the warmer temperatures (=28 °C)

associated with Fusarium wilt (Jones et al., 1991). Millipore-filtered bi-distilled water

(0.05 pm, Elgastat© Ultra High Polishing unit, O. Kleiner AG. Wohlen, Switzerland)

was added as needed to maintain a solution level of 1—2 cm. Fourteen days after

planting, tomato seedlings were carefully removed from the rockwool with the upper 1.5

cm of the root system attached. Fresh plant weight was measured and disease severity

was rated on a scale of 0 to 4 (adapted from Mihuta-Grimm et al., 1990) where

0=symptomless; l=slight brown discoloration of the upper root system; 2=moderate

brown discoloration of two-thirds or less of the upper root system; 3=extreme brown

discoloration of the upper root system and numerous necrotic lesions extending up the

crown and stem; 4=seedling dead or nearly so. Representative samples of necrotic tissue

were plated onto Komada's selective medium (Komada, 1975) to confirm F. oxyspormn

f.sp. radicis-lycopersici as the cause of symptoms.

Mineral treatments were used at seven rates, each consisting of three replicates

over time with 12 to 20 plants per replicate. Data for each mineral treatment were

analyzed separately. Relationships between amendment rate and disease severity and

plant weight were evaluated using regression procedures (SAS Institute Inc., Cary, NC,

USA).

106

Results and Discussion

Twelve mineral amendments were tested separately for their influence on Fusarium

crown and root rot. A similar and moderate level of disease ( 1—-2 rating on a scale of 4)

developed in all control treatments that were provided with the standard nutrient

solution (no additional minerals) (Figs. 1—3). Disease developed rapidly in affected

seedlings with necrotic lesions forming at the crown generally within a week of

emergence. This was often followed by severe chlorosis and turgor loss. No signs of

vascular infection were observed beyond the area of superficial necrosis. In treatments

provided with certain mineral amendments, severe disease developed (mean rating

3—4) and affected seedlings often failed to emerge. Seedlings with no symptoms after

10 days usually remained healthy even when left to mature (data not shown), probably

because of poor spread of the pathogen in rockwool (Mihuta-Grimm et al. 1990). In all

treatments, fresh weight of tomato seedlings was inversely correlated with the severity

of Fusarium crown and root rot (Figs. 1—4) supporting previous observations for this

disease (Duffy and Défago, 1997; Jones et ab. 1991; Woltz et ak, 1992). Windborne and

insect-transmitted spores were not a factor in this study because non-inoculated plants

that were placed in the greenhouse (but not included in the experiments) remained

disease-free.

Nitrogen form had a major influence on the severity of Fusarium crown and root

rot and on growth of tomato seedlings in a soilless rockwool system (Fig. 1). Increasing

concentrations of nitrate-N reduced disease and improved plant growth compared to

ammonium forms. Ammonium sulphate and ammonium chloride gave similar responses

implicating NH/ as the nocuous component (Fig 1). Ammonium nitrate has been

reported to act the same as ammonium-N or to have no influence on Fusarium diseases

107

(Jones et al. 1989; Schneider, 1985). While this was generally truc for Fusarium crown

and root rot, by testing a range of concentrations we were able to observe that at

concentrations below 100 mg N/liter disease was reduced in a fashion similar to nitrate-

N. The negative influence of the NH," ion was evident only at higher concentrations.

4

3cn<z

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£ 1

CD> 0CD</>

CDÜ) 4COCD

Î2 3

b

2

1

0

0 100 200

0 100

0 100

0 100 200

500

400

400 500

N (mg/liter)

350

^00E

251'

200

150

*\I .. y=502 1-70 9Ln(x)

Ad] r'=0 54

"00

30

*

I

CD

E,

ZzCO

CD

to

CD

c

_Ç0

350

3C0

2^0

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•50

100

50

0

XF

y^519 7-76 5Ln(x)Ad] rM)77

i^-| ~~~—"^—*—-—E

100 200 300 400

100 200

N (mg/liter)

Figure 1. Influence of nitrogen amount and form on Fusarium crown and root rot severity and

on tomato seedling growth after 2 weeks. Nitrogen was supplied at planting as (NH4),S04, (A,

E); NHC1, (B, F); Ca(NO ),4RO, (C, G): and NH NO. (D. H). Values represent the means

per plant in three trials. Adjusted regression coefficients and line derivations for were

significant at P = 0.0001, except for D and H where P = 0 0005 and 0.0125, respectively.

Vertical bars represent ± standard error of the mean.

108

It may be that at higher concentrations, seedlings had insufficient available

carbohydrates to convert the excess ammonium, which is toxic to tomato seedlings

(Woltz ct ak. 1992), to non-toxic amino acids (Pate, 1973).

Nitrates have long been recognized for reducing seedling disease caused by

Rhizoctonia solani (Huber and Watson, 1974); however, contradictory results have been

reported for Fusarium crown and root rot. Mihuta-Grimm et al. (1990) reported that

nitrogen supplied as a 20-20-20 (NPK) fertilizer had no effect on growth of F.

oxysporum f.sp. radicis-lycopersici in rockwool compared to non-fertilized treatments.

Jarvis and Thorpe (1980) found no effect of nitrogen form (total N applied was not

specified) on disease or yield when adequate lime was provided to negate potential pH

effects. Indeed, a differential effect of nitrogen form on pH is a major mechanism of

action for suppression of many soilborne pathogens (Huber and Watson. 1974). In

contrast, Woltz et al. (1992) reported that nitrate- vs. ammonium-N (each at 225 mg

N/liter) reduced severity of crown and root rot without affecting soil pH. Similarly, we

observed no effect of nitrogen fertilization (or any other mineral amendment) on pH of

the hydroponics solution. In both studies though, only pH of the bulk media was

measured and possible localized pH effects in the rhizosphere where disease actually

occurs cannot be excluded (Smiley, 1975). Another possible factor may be total calcium

concentration, which increases with liming and with calcium nitrates as used in our

study and that of Woltz et al. (1992). They found that all treatments that increased

calcium content also reduced disease. While adequate calcium in tomato tissues appears

to be important for resistance to cell-wall-degrading enzymes (e.g., polygalacturanase)

produced by Fusarium spp., it is considered a minor factor in controlling disease (Jones

etak, 1989).

109

Sodium phosphate increased disease severity and reduced tomato growth (Fig. 2).

Similar results with various phosphorus forms have been reported for wilt diseases

caused by related formae speciali (Jones et ak, 1989) and this has been attributed in

c

CO

Q 0^

y=0 06Ln(x)-0 48

Ad] r:=0 6344

100 200 300 400 500 100 150 200 250

o> 350-

ë- 300B

y=388 4-49 6Ln(x)

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300'D

D)250-

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r. 15a

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150'

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•

£ 1001 x ^~*~~^——«—-__ 100

c 50-(O

1 I 501

a.^

3 100 200 300 400 500 0 50 100 150 200 250

P(mg/liter) Mg (mg/liter)

Figure 2. Influence of sodium-phosphate (A, B) and magnesium-sulphate (C, D) on Fusarium

crown and root rot severity and on tomato seedling growth after 2 weeks. Values represent the

means per plant in three trials. Adjusted regression coefficients and line derivations for A-B

were significant at P < 0.0009. Differences in C-l) were not significant (P > 0.6000). Vertical

bars represent ± standard error of the mean.

part to the enhanced uptake of calcium (Jarvis, 1992). While possible effects of sodium

cannot be excluded, exhaustive studies with other Fusarium diseases [i.e., celery

yellows (Schneider, 1985) and asparagus wilt (Elmer, 1992; 1995)] found no influence

of the Na2' ion. Magnesium sulphate fertilization had absolutely no effect on Fusarium

crown and root rot at concentrations up to 250 mg Mg/liter (Fig. 2). To our knowledge,

this is the first examination of magnesium on F. oxysporum independent of the CI ion.

Previous work has demonstrated that fertilization with VlgCk increased wilt caused by

F. oxysporum formae speciali on tomato (Jones et ak, 1989) and celery (Schneider.

MO

1985). In these studies, Cl and not Mg2+ was identified as the problematic ion; a

conclusion which is further supported by our results.

Copper sulphate effectively reduced disease severity (Fig. 3) and improved tomato

growth (Fig. 4) at concentrations above 20 mg Cu/liter. This is not surprising

A

C

CO

CD>CD(/}

CDa)(0CDCO

Q

y=1 91-0 25Ln(x)

Ad) r2=0 67

Q0l-0

40 60

Cu (mg/liter)

y=0 73+0 34x

Adj r2=0 67

20 40 60 80

Zn (mg/liter)

100

y=0 99+0 25x

Adj r2=0 43

30 60 90 120

Mn (mg/liter)

20

y=0 63+0 44Ln(x)

Ad) r2=0 82

40 60 80 100

Mo (mg/liter)

y=1 26+0 41ln(x)

Adj r?=0 74

40 60 80 100

Mo (mg/liter)

y=1 08+0 31Ln(x)

Ad] r2=0 59

150 0 20 40 60 80 100

Fe (mg/liter)

Figure 3. Influence of micro-nutrients on the se\enty of Fusarium crown and loot lot of

tomato. Amendments of A, CuSO, 5H O. B, ZnS04 7RO; C. MnS04; D, MoO ; E,

(NHl)6Mo70,44ITO. and F, Fe-EDDHA were provided at planting. Values reptesent the means

per plant in three trials. Adjusted regression coetficients and line derivations were significant at

P < 0.0008. Veitical bars represent 2 standaid error of the mean

ill

considering the historical use of Cu2+ as a broad-spectrum fungicide (Schumann, 1991 ).

No phytotoxicity was observed even at the highest concentrations tested. In contrast, all

other micronutrients tested aggravated disease (Fig. 3) and stunted growth (Fig. 4). Zinc

and manganese increased disease at concentrations above 40 and 50 mg/liter,

respectively. Molybdenum increased disease at concentrations of 20 mg/liter and above.

Ammonium molybdate (Fig. 3E) was more conducive than molybdate (Fig. 3D),

perhaps because of the additive influence of the Nil, ions. Iron added as Fe-EDDFIA

increased disease at concentrations of just 5 mg/ml and greater, with only slight further

increases until over 80 mg/ml (Fig. 3F). We found no evidence for induction of host

defenses by zinc, iron or manganese as suggested by Mandai and Sinha (1992). Plants

supplied with zinc or ammonium molybdate at 33 mg/liter exhibited no signs of toxicity

in the absence of the pathogen (Duffy and Défago. 1997). At higher concentrations

though, we observed that node length was increasingly shortened, suggesting that

phytotoxicity as well as increased disease possibly contributed to reductions in fresh

weight (Fig. 4). While excessive concentrations are uncommon in agricultural soils and

hydroponics, their availability can be increased under certain conditions such as

acidification. Incidentally, acidic pH also favors crown and root rot (Woltz et al.. 1992).

Inert materials such as rockwool tend to reduce plant sensitivity to minerals (measured

as high electrical conductivity) (Jarvis, 1992). Plants may be exposed to elevated

mineral concentrations applied to improve the beneficial activity of biological control

strains of Pseudomonas fluorescens (Duffy and Défago. 1997). Results of our study

facilitate the development of such microbe-mineral treatments with minimal adverse

side effects on the host plant, information that has been lacking. It also accentuates the

need to develop methods for more efficient delivery of potentially phytotoxic minerals.

112

o 300" T J300

E

î»~ 250- 250_c

CT>

03 20°

y=225 5+9 92Ln(x)200

JC 150 150m Adj r"=0 14

i 100 100

+~t

S 50

Û.A

50"

0 1 1

0 20 40 60 80 100

Cu (mg/liter)

S 3004 300

E frT^T 250jf«-^J y=287 0 28 Ox 250-

§>200 J Adj r =0 62 200

4=150 150

CO

E ioo1 ^^"^^--^ 100"

i+_

«50'

B50-

o-

20 40 60

Zn (mg/hter)

100

y=308 4-27 8x

Adj r?=0 69

60 90

Mn (mg/hter)

D

y=268 1 23 8x

Adj r2=0 44

40 60 80

Mo (mg/hter)

100

o : 0 40 60 80 100

Mo (mg/liter)

300

250 L_^ T

200l

150

100 y=268 8 20 7x

50

pAdj r2=0 27

0 I ii

0 20 40 60 80 100

Fe (mg/liter)

Figure 4. Influence of micro-nutiients on tomato seedling giowth aitei 2 weeks in îockwool

infested with / usanum owspoium t sp îaduis hcopctsn i Amendments of A, CuSO, 5H O,

B, ZnS04 7H O, C, MnSO .D, MoO E, (NFL) Mo O

44H O and F, Fe-FDDHA weie

piovided at planting Values lepiesent the means pei plant m thiee tnals Adjusted îegiession

coefficients and line demations wete signitieant at F < 0 0007 toi all except A and F where P

= 0 0504 and 0 0091, tespeetneh Vertical bais lepiesent + standaid enoi of the mean

113

Exactly how minerals influence disease is uncertain but effects on pathogen

activity and host susceptibility are likely involved. Fusarium oxysporum has a relatively

high requirement for micronutrients (Jones et ak. 1989). Concentrations of

zinc, iron, manganese and other metals above those typically found in soil solutions

stimulates growth and sporulation (Duffy and Défago, 1997; Jones et ak, 1989). The

profile of secondary metabolites produced by the pathogen, including phytotoxic

compounds like fusaric acid, is also altered (Duffy and Défago. 1997; Egli, 1969).

Nitrates inhibit both sporulation and spore germination while ammonium has the

opposite effect (Jones et ak. 1989). Susceptibility of tomato to fungal attack is increased

by zinc partly because it raises sugar status in plant tissues (Jarvis, 1992). Host

susceptibility can also be altered by interactions between minerals, particularly at

elevated concentrations, which impact nutrient availability. For example, ammonium-N

interferes with the uptake of nitrates and potassium which in turn stimulates chloride

uptake leading to increased susceptibility of tomato to F. oxyspormn f.sp. lycopersici

(Jarvis, 1992). Disease suppression with mineral nutrients has also been attributed to the

stimulation of indigenous populations of antagonistic micro-organisms in the soil and

rhizosphere (Elmer, 1995; Engelhard, 1989). While this is generally not relevant in

hydroponics, recent work indicates that mineral nutrients can be exploited to improve

the beneficial activity of introduced biocontrol agents (Duffy and Défago, 1997).

Our results build on those of Woltz et al. (1992) and provide a foundation for

developing a control strategy based on plant nutrition. Such an approach has been

successfully applied to manage other Fusarium diseases (Jarvis. 1992; Jones et ak,

1989). Mineral effects on crown and root rot of tomato caused by F. oxysporum f.sp.

radicis-lycospersici were similar to what has been reported for other formae speciali

114

which reflects the adaptability of control strategies for various Fusarium diseases. It

further suggests biological similarity of these pathogens and/or similar responses of

diverse hosts to these fungi. Fusarium crown and root rot. however, is not the only

problem threatening hydroponically-grown tomato and non-target effects of certain

amendments on other diseases need to be considered. A prominent example, nitrate-N

which reduced crown and root rot has the opposite effect aggravating economically

devastating diseases caused by Pythium and phytopathogenic bacteria (Stanghellini and

Rasmussen, 1994). Integrating biocontrol agents and/or fungicides at reduced non-

phytotoxic concentrations with mineral amendments may enhance the level and

spectrum of disease control.

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Sivan, A., O. Ucko, and I. Chet. 1987. Biological control of Fusarium crown rot of tomato by

Triehoderma harzianum under field conditions. Plant Dis. 71:587-592.

Smiley, R. W. 1975. Forms of nitrogen and the pH in the root zone and their importance to root

infections, p. 55-62. In: G. W. Braehl (ed.). Biology and control of soil-borne plant

pathogens. The American Phytopathological Society Press. St. Paul, MN, USA.

Stanghellini, M. E., and S. L. Rasmussen. 1994. Hydroponics: a solution for zoosporic

pathogens. Plant Dis, 78:1129-1138.

Woltz, S. S., J. P. Jones, and J. W. Scott. 1992. Sodium chloride, nitrogen source, and lime

influence Fusarium crown rot severity in tomato. HortScience 27:1087-1088.

Ziegler, H. 1983. Die Nährstoff und ihr Umsatz in der Pflanze. 2. Verfügbarkeit der

Nährelement, p. 334-336. In: E. Strassburger, F. Noll. II. Schenk, and A. F. W. Schimper

(eds.). Lehrbuch der Botanik. Gustav Fischer Verlag. Stuttgart and New York.

117

General Conclusions

Pseudomonas fluorescens inoculants are widely used in agriculture as soil and seed

treatments to suppress plant diseases caused by soilborne fungi, so-called biocontrol.

However, variable performance in different environments (eg., among sites and between

cropping seasons) has been a major obstacle to commercialization of biocontrol

products. Tackling this problem requires an understanding of the environmental factors

that influence the activity of biocontrol strains, particularly factors that affect key

biocontrol processes like antibiotic biosynthesis. Such information has been lacking.

The overall objective of this thesis was to identify environmental factors that influence

the biocontrol activity of P. fluorescens. Focus was placed on minerals and pathogen

signals because these are commonly encountered by biocontrol pseudomonads in the

environment.

Antibiotic production is regulated by the membrane-bound sensor kinase gacS

and the transcriptionally activated response regulator gacA. Chapter 1 reports that these

genes spontaneously mutate at an extremely high frequency under standard culture

conditions which are used to mass-produce inoculants. Mutation was found to reduce

biocontrol activity of inoculants. Mutant accumulation was reduced by amending

culture media with certain trace-minerals (eg., copper, zinc, ammonium-molybdate,

manganese) or by using media with reduced nutrient concentrations. This result has

important applications for other bacterial strains as a simple, cost-effective way improve

genetic stability and improve inoculant quality. This work may also have important

relevance for plant and human pathogenic pseudomonads, in which gacS-gacA regulate

production of virulence metabolites.

Antibiotic production is a primary mechanism by which most biocontrol strains

suppress plant disease. Chapter 2 reports that certain minerals (eg., zinc and ammonium-

molybdate) also stimulate biosynthesis of various antibiotics in a strain dependent

fashion. This may also point the way to devlopment of a bioassay for heavy-metal

detection in environmental sampling. Such an approach has been taken with siderophore

biosynthesis and quantification of iron in soil and rhizosphere environments (Loper and

Henkels 1997).

118

Pathogens have been known to influence the growth and biocontrol activity of

Pseudomonas strains, but the mechanism for this has never been investigated. Chapter 3

reports that fusaric acid produced by Fusarium oxysporum f.sp. radicis-lycopersici

blocked bacterial antibiotic production and interefered with biocontrol of tomato root

disease. Zinc improved biocontrol in a hydroponic system because it repressed fusaric

acid production by the pathogen, thereby creating a more favorable environment for the

biocontrol agent. Chapter 4 extended this work using an ecologically and genetically

diverse collection of strains to show that biocontrol activity againt tomato crown and

root rot was indeed negatively correlated with fusaric acid sensitivity. Thus sensitive

strains like CHAO are relatively ineffective. Resistant strains (eg., Q2-87) exist though

and these are more effective.

Together the work with minerals and fusaric acid will refine strain

selection. Just because a strain has the genes to produce an antibiotic like PHL doesn't

mean it produces it in all environments. It may be possible to identify strains that are

more likely to work in particular environments, so called 'prescription biocontroF.

Strain mixtures may give a more reliable control under vacilating environmental

conditions. Genetically modifying strains to relieve key genes from environmental

signal control may be a viable way to improve biocontrol. On the other hand, using

mineral amendments to improve biocontrol may be less controversial than

biotechnological approaches. Chapter 4. though cautions that even minerals can have

unexpected side-effects and that any amendments should consider potential adverse

impact such as increased disease or inhibition of other beneficial microorganisms.

Loper, J. E., and M. D. Henkels. 1997. Availability of iron to Pseudomonas fluorescens in

rhizosphere and bulk soil evaluated with an ice nucleation reporter gene. Appl. Environ.

Microbiol. 63:99-105.

119

Acknowledgments

There are so many people to thank for so much that I think it 2s finally appropriate to use

my favorite quote from an Oscar2s acceptance speech. In 198 L Maureen Stapleton. Best

Supporting Actress for 'Reds2 clutching her little gold man and weeping hysterically,

'I thank everyone that I have ever met in my entire life'

So THANKS... everyone... with especial thanks to Geneviève Défago, Ruth Duffy.

David Weller, Tili Rosenberger, my Phytomed comrades... and Stefan

120

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121

Curriculum vitae

Brion DUFFY

Bom August 21,1967

1987-88 Research Assistant, Nitrogen Fixation in Tropical Agricultural Legumes

Project (NifTAL), Univ of Hawaii Manoa (BB Bohlool, PS Singleton)

1988 BSc, Crop Protection, Univ of Hawaii Hilo (JA Fernandez)

1988-89 Research Assistant, Soil Microbiology, EMBRAPA, Rio de Janeiro, Brasil

(J Döbereiner, RM Boddey)

1992 MSc, Dept Plant Pathology, Washington State Univ (DM Weiler)

1992-93 Research Plant Pathologist, Hawaii Volcanoes National Park, CooperativeParks Studies Unit, Univ of Hawaii Manoa (DE Gardner, C Smith)

1992-93 Lecturer, Univ of Hawaii Hilo, College of Agriculture (J Fujii)

1994 PhD candidate, Institute of Plant Sciences, Swiss Federal Institute of

Technology Zürich ETH-Z (G Défago)

' ^i^aT:«a» ,/ **.**

i

m

Publications

1. Duffy, B. K. Field survival of the anthunum blight pathogen Xanthomonas

campestns pv. dieffenbachiae in crop residues. Eur. J. Plant Pathol., submitted.

2. Duffy, B. K., and Gardner, D. E. 1999. Nematodes associated with the invasive

weed, Mynca faya, in Hawaii. Nematropica, in press June '99.

3. Duffy, B. K., and Défago, G. 1999. Environmental signals modulate antibiotic and

siderophore production by Pseudomonas fluorescens biocontrol strains. Appl.Environ. Microbiol., in press June '99.

4. Duffy, B. K., and Défago, G. 1999. Micro- and macroelements influence the

severity of Fusarium crown and root rot of tomato in a soilless production system.

HortScience, in press April '99.

5. Duffy, B. K., and Défago, G. 1997. Zinc amendment improves the biocontrol of

tomato crown and root rot by Pseudomonas fluorescens and represses the

production of pathogen metabolites inhibitory to bacterial antibiotic biosynthesis.

Phytopathology 87: 1250-1257.

6. Duffy, B. K., Ownley, B. H., and Weiler, D. M. 1997. Soil chemical and physical

properties associated with suppression of take-all of wheat by Frichoderma

komngn. Phytopathology 87:1118-1124.

7. Duffy, B. K., Simon, A., and Weiler, D, M. 1996. Combination of Frichoderma

komngn with fluorescent pseudomonads for control of take-all on wheat.

Phytopathology 86:188-194.

8. Duffy, B. K., and Weiler, D. M. 1996. Biological control of take-all of wheat in the

Pacific Northwest of the USA using hypovirulent Gaeumannomyces graminis var.

tntici and fluorescent pseudomonads. J. Phytopathol 144-585-590.

9. Duffy, B. K., and Weiler, D. M. 1995. Suppression of take-all of wheat using

Gaeumannomyces graminis var. graminis individually and in combination with

fluorescent Pseudomonas spp. Plant Dis. 79'907-911.

10. Duffy, B. K., and Gardner, D. E. 1994. Locally established Botrytis fruit rot of

Mynca faya, a noxious weed in Hawaii. Plant Dis. 78'919-923.

11. Duffy, B. K., and Weiler, D. M. 1994. A new semiselective and diagnostic medium

for Gaeumannomyces graminis var tntici Phytopathology 84:1407-1415.

Minor journals, Books, Technical reports

1. Duffy, B., Rosenberger, U., and Défago, G., eds. 1998. Molecular Approaches in

Biological Control IOBC wprs Bull. 21(9). 324 p.

2. Duffy, B.K., and Defago, G. 1998. A Fusarium pathogenicity factor blocks

antibiotic biosynthesis by antagonistic pseudomonads IOBC wprs Bull. 21(9):145-148.

3. Duffy, B. K. 1997. Susceptibility of Chinese and Cuban rice cultivars to blast,sheath blight, and bacterial blight. Ann. Appl. Bio!, 130 (Supplement), Tests

Agrochem. Cult. 18 40-41

4. Duffy, B.K., and Defago, G 1997. Environmental signals in biocontrol. p. 421-425.

In: Plant Growth-Promoting Rhizobacteria - Present Status and Future Prospects.A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N Kondo, and S. Aikino, eds.

OECD, Pans

5. Duffy, B.K., and Défago, G. 1997. Environmental signals in biocontrol of tomato

root disease by Pseudomonas fluorescens. Med. Fac Landbouww. Univ. Gent

62/3b:1015-1022.

6. Duffy, B.K. 1996. Helping non-native English speakers Publish: Why and How?

Phytopathol. News 7 111

124

7. Duffy, B.K., and Gardner, D.E. 1995. Decline of invasive faya in Hawaii Newsl.

Haw. Bot. Soc. 34:1-5.

8. Défago, G., and Duffy, B.K. 1994, Wellington & van Elsas' 'Genetic interactions

among microorganisms in the natural environment'. Eur. J. For. Pathol. 24:64.

9. Duffy, B.K., and Gardner, D.E. 1993. Phytopathogenic fungi associated with fruits

of pukiawe (Styphelia tameiameiae). Newsl. Haw. Bot. Soc. 32:6-8.

10. Duffy, B.K., and Gardner, D.E. 1993. Dieback of mamane (Sophora chrysophylla)and rat depredation. Newsl. Haw. Bot. Soc. 32:8-13.

11. Fernandez, J.A., Tanabe, M.J., Moriyasu, P., Duffy, B.K. 1989. Biological control.

p. 27-29. In: Proc. 2nd Anthurium Blight Conference. J.A. Fernandez and W.T.

Nishijima, eds. Univ. of Hawaii at Hilo, Hawai'i.

12. Fernandez, J.A., Tanabe, M.J., Duffy, B.K. 1988. Biological control. In: Proc. 1st

Anthurium Blight Conference. Univ. of Hawaii at Hilo, Hawai'i.

13. Duffy, B.K., and Gardner, D.E. Etiological investigations of the mortality of invasive

Myrica faya in Hawai'i. Univ. Hawaii Coop. Park Stud. Unit Tech. Rep., in press.

14. Yang, P., Duffy, B.K., Gardner, D.E., and Foote, D. Inventory of herbivorous

insects on fire tree, Myrica faya, in Hawai'i. Univ. Hawaii Coop. Park Stud. Unit

Tech. Rep., in press.